CN101668856B - Optimized non-canonical zinc finger proteins - Google Patents

Abstract

Disclosed herein are zinc fingers comprising CCHC zinc coordinating residues. Also described are zinc finger proteins and fusion proteins comprising these CCHC zinc fingers as well as polynucleotides encoding these proteins. Methods of using these proteins for gene editing and gene regulation are also described.

Description

Through the non-standard zinc finger protein for optimizing

Cross-reference to related applications

This application claims the U.S. Provisional Application No.60/874,911 of the submission of on December 14th, 2006 and May 30 in 2007 The U.S. Provisional Application No.60/932 that day submits to, 497 rights and interests completely include both of which disclosures content herein by referring to.

Invention field

Present disclosure is in genome project (genome engineering), gene targeting (genetargeting), target To chromosomal integration (targeted chromosomal integration), protein expression (protein expression) In exopathogenic factor genome editor (epigenome editing) field.

Background of invention

Protein is combined to the sequence-specific of DNA, RNA, protein and other molecules and is related to many cell processes, such as Such as transcription, duplication, chromatin Structure, restructuring, DNA reparations, RNA processing and translation.Participate in protein-DNA, protein-RNA Contribute to developing with the binding specificity of the cell binding protein of protein protein interaction, break up and homeostasiss.

Zinc finger protein (ZFP) is the protein that can combine DNA with sequence-specific fashion.Zinc finger is initially from African pawl Toad (African clawed toad) be Xenopus laevis oocytes transcription factor TFIIIA in identify.It is this kind of The single zincfinger motif domain of ZFP is about 30 aminoacid, and several structural researches have been proven that it (is included comprising β-corner Two conservative cysteine residues) and alpha-helix (including two conservative histidine residues), they are via two and half Guang ammonia Acid and two histidine coordinated Zn atoms and keep specific conformation.This kind of ZFP is also referred to as C2H2 ZFP.The ZFP of other type There is prompting.See, for example, the Jiang etc. (1996) discussed with regard to Cys-Cys-His-Cys (C3H) ZFP J.Biol.Chem.271：10723-10730.So far, identifying known to thousands of kinds or in the transcription factor of presumption Go out more than 10,000 kinds of zinc finger sequences.Zinc finger domain be not only involved in DNA identification, but also participate in RNA combine and protein-egg White matter is combined.Estimate that this quasi-molecule can account for about the 2% of all human genes at present.

Most of zinc finger proteins have conservative cysteine and histidine residues, their four sides in each finger domain The single zinc atom of bodily form ground coordination.Specifically, most of ZFP are with general sequences as-Cys- (X)_2-4-Cys-(X)₁₂-His- (X)_3-5-His-(SEQ ID NO：1) finger component is characterized, and wherein X represents any aminoacid (C2H2ZFP).It is this by most wide The zinc coordination sequence of the type of general presentation includes two cysteine with specific distance and two histidine.Each folding for referring to Stack structure includes antiparallel β-corner, finger tip area and short amphipathic alpha-helix.Metal-coordinated ligand combines zinc ion, and And in the case of zif268 type zinc fingers, short, amphipathic alpha-helix is combined in the major groove of DNA.In addition, the structure of zinc finger is led to Cross some conservative hydrophobic amino acid residues (such as the residue and helical segments in referring to just before first conservative Cys+ The residue of 4) and stabilisation is obtained by the zinc coordination of conservative cysteine and histidine residues.

Produce the contact of direct base position, close to base contacting positions " supportive " or " supportive " residue and Specification (C2H2) zinc finger protein that there is change in the position of DNA phosphate backbones can be contacted to have been described.See, for example, United States Patent (USP) No.6,007,988；6,013,453；6,140,081；6,866,997；6,746,838；6,140,081；6,610, 512；7,101,972；6,453,242；6,785,613；7,013,219；PCT WO 98/53059；Choo etc. (2000) Curr.Opin.Struct.Biol.10：411-416；Segal etc. (2000) Curr.Opin.Chem.Biol.4：34-39.

In addition, the zinc finger protein comprising the zinc finger that residue is coordinated with improved zinc also has been described and (see, for example, the U.S. Patent application No.20030108880；20060246567；With 20060246588；The disclosure of which is included by referring to).So And, although the zinc finger protein comprising these non-standard zinc fingers remains gene transcription regulation function, but they play zinc finger nucleic acid The ability of enzyme (ZFN) effect decreases in some cases relative to the zinc finger protein being made up of specification C2H2 zinc finger only.

So, it is still necessary to which (particularly in the structure of Zinc finger nuclease) includes the non-standard zinc coordination having through optimization Other through engineering approaches zinc finger associated proteins of the zinc finger in area.

Summary of the invention

This disclosure provides having the zinc finger dna binding structural domain for changing at least one zinc is coordinated residue.Tool Body ground, there is described herein CCHC zinc fingers.These CCHC zinc fingers further can be coordinated near residue (for example in zinc finger most in zinc In residue around (C-terminal-most) zinc coordination residue of C-terminal) (substitute, insert and/or delete comprising other change Except).Also describe the zinc finger polypeptide comprising one or more of these CCHC zinc fingers and fusion protein, encode these zinc fingers and fusion Polynucleotide of albumen and using these zinc finger polypeptides and/or the method for fusion protein.

Thus, the embodiment that present disclosure is covered but is not limited to lower column number：

1. a kind of zinc finger protein, it includes non-standard (non-C₂H₂) zinc finger, wherein the non-standard zinc finger has involves DNA With reference to spiral part and wherein described spiral part zinc co-ordination area include aminoacid sequence HX₁X₂RCX_L(SEQ ID NO： 2)；And wherein described zinc finger protein is transformed into and combines target sequence.

2. the zinc finger protein of embodiment 1, wherein X₁It is A, and X₂It is Q.

3. the zinc finger protein of embodiment 1, wherein X₁It is K, and X₂It is E.

4. the zinc finger protein of embodiment 1, wherein X₁It is T, and X₂It is R.

5. the zinc finger protein of embodiment 1, wherein X_LIt is G.

6. a kind of zinc finger protein, it includes two or more zinc fingers, and wherein at least one zinc finger includes sequence C ys- (X^A)_2-4-Cys-(X^B)₁₂-His-(X^C)_3-5-Cys-(X^D)_1-10(SEQ ID NO：3), wherein X^A、X^B、X^CAnd X^DCan be any Aminoacid.

7. the zinc finger protein of any one of embodiment 1 to 6, its include table 1, table 2, table 3 or table 4 it is arbitrary shown in arbitrary sequence Row.

8. the zinc finger protein of embodiment 6 or 7, wherein X^DComprising sequence QLV or QKP.

9. the zinc finger protein of embodiment 8, wherein sequence QLV or QKP be the zinc finger 3 C-terminal aminoacid it is residual Base.

10. the zinc finger protein of any one of embodiment 6 to 9, wherein X^DComprising 1,2 or 3 Gly (G) residue.

A kind of 11. zinc finger proteins, it includes multiple zinc fingers, and wherein at least one zinc finger is included according to embodiment 1 to 10 The CCHC zinc fingers of any one.

The zinc finger protein of 12. embodiments 11, wherein the zinc finger protein includes 3,4,5 or 6 zinc fingers.

The zinc finger protein of 13. embodiments 11 or 12, its middle finger 2 includes the CCHC zinc fingers.

The zinc finger protein of any one of 14. embodiment 11 to 13, wherein C-terminal zinc finger refer to comprising the CCHC.

The zinc finger protein of any one of 15. embodiment 11 to 14, wherein at least two zinc finger includes the CCHC zinc fingers.

The zinc finger protein of any one of 16. embodiment 11 to 15, wherein the zinc finger protein includes arbitrary sequence shown in table 8 Arrange and be transformed into reference to the target sequence in IPP2-K genes.

A kind of 17. fusion protein, it includes the zinc finger protein and one or more functions of any one of embodiment 1 to 16 Domain.

A kind of 18. fusion protein, it is included：

(a) cutting half domain (half-domain),

The zinc finger protein of any one of (b) embodiment 1 to 16, and

ZC joints between (c) insertion cutting half domain and the zinc finger protein.

The fusion protein of 19. embodiments 18, wherein the length of the ZC joints is 5 aminoacid.

The fusion protein of 20. embodiments 19, wherein the aminoacid sequence of the ZC joints is GLRGS (SEQ ID NO： 4)。

The fusion protein of 21. embodiments 18, wherein the length of the ZC joints is 6 aminoacid.

The fusion protein of 22. embodiments 21, wherein the aminoacid sequence of the ZC joints is GGLRGS (SEQ ID NO： 5)。

A kind of 23. polynucleotide, its coding is according to the zinc finger protein of any one of embodiment 1 to 16 or according to embodiment The fusion protein of 17 to 22 any one.

24. is a kind of for the chromatinic method of targeting incising cell in plant cell, and methods described is included in described thin A pair fusion protein according to any one of embodiment 18 to 22 are expressed in born of the same parents, wherein：

(within tennucleotides within target sequence 10 nucleotide apart of (a) described fusion protein of each other)；And

(b) described fusion protein dimerization, and cut the DNA between the target sequence.

A kind of 25. methods of the targeting genetic recombination in host plant cell, methods described includes：

A () expresses a pair fusion protein according to any one of embodiment 18 to 22, wherein institute in the host cell The target sequence for stating fusion protein is present in selected host target genes seat；And

B () identification shows the recombinant host cell of sequence change in the host target genes seat.

The method of 26. embodiments 24 or 25, wherein it is the mutation being selected from the group that the sequence changes：Hereditary material is deleted Except, the insertion of hereditary material, the replacement of hereditary material and its any combinations.

The method of any one of 27. embodiment 24 to 26, it further includes for exogenous polynucleotide to import the host In cell.

The method of 28. embodiments 27, wherein the exogenous polynucleotide is comprising homologous with the host target genes seat Sequence.

The method of any one of 29. embodiment 24 to 28, wherein the plant is selected from the group：It is monocotyledon, dicotyledonous Plant, gymnosperm and Eukaryotic Algae.

The method of 30. embodiments 29, wherein the plant is selected from the group：Semen Maydiss, rice, Semen Tritici aestivi, Rhizoma Solani tuber osi, Semen sojae atricolor, kind Eggplant, Nicotiana tabacum L., brassicaceae (Brassica family) member and Arabidopsises (Arabidopsis).

The method of any one of 31. embodiment 24 to 29, wherein the plant is tree.

The method of any one of 32. embodiment 24 to 31, wherein the target sequence is in IPP2K genes.

33. it is a kind of for reducing seed in Phytic Acid Levels method, methods described include being inactivated according to embodiment 32 or Change IPP2-K genes.

34. is a kind of for making phosphorus that the method for utilizing can be more metabolized in seed, and methods described is included according to embodiment 32 inactivations change IPP2-K genes.

A kind of 35. plant cells, it includes zinc finger protein according to any one of embodiment 1 to 16, according to embodiment The fusion protein of 17 to 22 any one or the polynucleotide according to embodiment 23.

The plant cell of 36. embodiments 35, wherein the cell is seed.

The plant cell of 37. embodiments 36, wherein the seed is corn seed.

The plant cell of any one of 38. embodiment 35 to 37, wherein IPP2-K is partially or completely inactivated.

The plant cell of 39. embodiments 38, wherein the Phytic Acid Levels in the seed are to reduce.

The plant cell of 40. embodiments 35 to 39, wherein the metabolism of the phosphorus in the cell can be to improve using level 's.

Brief description

Fig. 1 be depicted in United States Patent (USP) No.2005/0064474 and GFP Cell Reports measurement system described below with The figure of the intergenic suppression rate (gene correction rate) of the percentage ratio measurement of the cell of expression GFP.ZFN variants are referred to as " X-Y, " wherein " X " refers to table number, and " Y " shows the numbering for giving the zinc finger in concrete selected table.For example, " 2-21 " refers to have and includes Sequence shown in the 21st row is HAQRCGLRGSQLV (SEQ ID NO in table 2：53) ZFN of finger.

Fig. 2 is described by the figure of the percentage ratio using various ZFN variants to carrying out cutting the Cel-1 signals for causing.It is logical Cross and consult the result that sample number into spectrum is that each pair ZFN shows two experiments.Show in the block for each sample in the upper right corner Variant pair, wherein " wt5-8 " and " wt5-9 " refers in the embodiment 14 (table 17) of U.S. Patent application No.2005/0064474 Specification ZFN pair of disclosure.In sample 3-12, replace the finger 2 of specification ZFN 5-8 or 5-9 with non-standard sequence or refer to 4 identification The C-terminal region of spiral.Above figure in the upper left corner shows sample 3-12 be referred to as 20,21,43,45,47 and 48 non-rule Finger position of the partial sequence and these variants of model ZFN variants in the ZFN of 4 fingers.Describe the result of experiment 2 of sample 8 and 9 Asterisk above cylindricality indicates the background in road, causes underestimating for ZFN effects.

Fig. 3 is the base in describing United States Patent (USP) No.2005/0064474 and GFP Cell Reports measurement system specifically described herein Because of the figure of corrected rate.ZFN pair tested in each sample is shown below each cylindricality, wherein zinc finger numbering 20th, 21,43,45,47 and 48 is that those are described in embodiment 3, and CCHC zinc fingers 1a to 10a includes sequence shown in table 3 and 4 Row.Zinc finger 20,21,7a, 8a, 9a and 10a used in Fig. 4；Zinc finger 43,45,47,48,1a used in Fig. 2,2a, 3a, 4a, 5a, And 6a.

Fig. 4 is a kind of linear diagram of plasmid pDAB1585 (targeting vector for Nicotiana tabacum L.).

Fig. 5 is a kind of diagram of plasmid pDAB1585 (targeting vector for Nicotiana tabacum L.)

Fig. 6 A and 6B depict Zinc finger nuclease (ZFN).Fig. 6 A are to describe the schematic diagram that ZFN is combined.Fig. 6 B show target The sequence of sequence.

Fig. 7 is the diagram of plasmid pDAB1400.

Fig. 8 is the diagram of plasmid pDAB782.

Fig. 9 is the diagram of plasmid pDAB1582.

Figure 10 is the diagram of plasmid pDAB354.

Figure 11 is the diagram of plasmid pDAB1583.

Figure 12 is the diagram of plasmid pDAB2407.

Figure 13 is the diagram of plasmid pDAB1584.

Figure 14 is the diagram of plasmid pDAB2418.

Figure 15 is the diagram of plasmid pDAB4045.

Figure 16 is the diagram of plasmid pDAB1575.

Figure 17 is the diagram of plasmid pDAB1577.

Figure 18 is the diagram of plasmid pDAB1579.

Figure 19 is the diagram of plasmid pDAB1580.

Figure 20 is the diagram of plasmid pDAB3401.

Figure 21 is the diagram of plasmid pDAB1570.

Figure 22 is the diagram of plasmid pDAB1572.

Figure 23 is the diagram of plasmid pDAB4003.

Figure 24 is the diagram of plasmid pDAB1571.

Figure 25 is the diagram of plasmid pDAB7204.

Figure 26 is the diagram of plasmid pDAB1573.

Figure 27 is the diagram of plasmid pDAB1574.

Figure 28 is the diagram of plasmid pDAB1581.

Figure 29 is the diagram of plasmid pDAB1576.

Figure 30 is the diagram of plasmid pDAB1600.

Figure 31 is the diagram of plasmid pDAB3731.

Figure 32 is the diagram of plasmid pDAB4322.

Figure 33 is the diagram of plasmid pDAB4331.

Figure 34 is the diagram of plasmid pDAB4332.

Figure 35 is the diagram of plasmid pDAB4333.

Figure 36 is the diagram of plasmid pDAB4334.

Figure 37 is the diagram of plasmid pDAB4336.

Figure 38 is the diagram of plasmid pDAB4339.

Figure 39 is the diagram of plasmid pDAB4321.

Figure 40 is the diagram of plasmid pDAB4323.

Figure 41 is the diagram of plasmid pDAB4341.

Figure 42 is the diagram of plasmid pDAB4342.

Figure 43 is the diagram of plasmid pDAB4343.

Figure 44 is the diagram of plasmid pDAB4344.

Figure 45 is the diagram of plasmid pDAB4346.

Figure 46 is the diagram of plasmid pDAB4330.

Figure 47 is the diagram of plasmid pDAB4351.

Figure 48 is the diagram of plasmid pDAB4356.

Figure 49 is the diagram of plasmid pDAB4359.

Figure 50 is the diagram of plasmid pDAB7002.

Figure 51 is the diagram of plasmid pDAB7025.

Figure 52 is the diagram of plasmid pDAB1591.

Figure 53 is the figure of plasmid pcDNA3.1-SCD27a-L0-Fok1 (for the DNA profiling that PCR expands Scd27ZFN) Show.

Figure 54 is the diagram of plasmid pDAB1594.

Figure 55 is the diagram of plasmid pDAB1598.

Figure 56 is the diagram of plasmid pDAB1577.

Figure 57 is the diagram of plasmid pDAB1578.

Figure 58 is the diagram of plasmid pDAB1601 (pat gene control vector).

Figure 59 is the schematic diagram of Intrachromosomal recombination for depicting prediction, being stimulated by IL-1-Fok1 fusion protein.

Figure 60 is the diagram of plasmid pDAB1590 (positive GFP expression control).

Figure 61 be depict prediction, the interchromosomal homologous recombination that stimulated by IL-1 zinc finger-Fok1 fusion protein shows It is intended to.

Figure 62 be depict prediction, the interchromosomal homologous recombination that stimulated by Scd27 zinc finger-Fok1 fusion protein shows It is intended to.

Figure 63 is the gel for depicting recombinant PCR analyses.Front 4 road in left side in gel overlay mark.The 1-5 of labelling Road shows the HR events of the BY2-380 conversions carried out come C3H IL-1-Fok1 antigen-4 fusion protein genes of using by oneself, the 6-7 of labelling Road shows the HR events of the BY2-380 conversions carried out come C3H SCD27-FokI antigen-4 fusion protein genes of using by oneself.

Figure 64 shows Semen Maydiss IPP2K gene orders (SEQ ID NO：6), it is derived from HiII cell cultures, and It serves as the design template of the through engineering approaches of the ZFN for targeting Semen Maydiss IPP2K.

Figure 65 (little figure A to E) depicts ZFN expression vector cloning approach.ZFN expression is produced using progressively Strategies For The Cloning Construct.Each ZFN encoding gene is cloned into into carrier pVAX-N2A-NLSop2-EGFP-FokMono (A) and pVAX-C2A- In NLSop2-EGFP-FokMono (B) with establishment binary protein box (dual-protein cassette) (C).The box is connected Access to generate final plasmid (E) in pDAB3872 (D), for expressing ZFN heterodimers.

The ZFN that Figure 66 is depicted in Semen Maydiss IPP2K genes is combined.The double-strand for needing two ZFN albumen to implement to DNA is cut Cut.Show sequence (the SEQID NO around cleavage site (with downward arrow instruction)：7).If a protein (8705) binding sequence CTGTGGGGCCAT (cochain) (SEQ IDNO：8), then another protein (8684,8685 or 8686) With reference to downstream sequence (CTTGACCAACTCAGCCAG, lower chain) (SEQ ID NO：9).

Figure 67 depicts wild type (top sequence, SEQ ID NO：10) 127 (bottom sequence, SEQ ID are cloned with ZFN NO：11) sequence.With the cutting target that grey box is highlighted the ZFN.

Figure 68 shows the non-of the dsDNA fractures by being mediated by ZFN in the detected Semen Maydiss IPP2K genes of 454 sequencings The comparison of various deletions that homologous end connection (NHEJ) causes.With the cutting target that grey box is highlighted the ZFN.

Figure 69 is depicted in United States Patent (USP) No.2005/0064474 and GFP Cell Reports measurement system specifically described herein In intergenic suppression rate figure.ZFN pair tested in each sample is shown below each cylindricality.

Figure 70 depicts the plasmid pDAB7471 built as described in embodiment 18B.

Figure 71 depicts the plasmid pDAB7451 built as described in embodiment 18C.

Figure 72 is the schematic diagram for depicting exemplary autonomous herbicide tolerance genes expression cassette.The structure such as embodiment 18D Complete promoter-transcript unit (PTU) is included as described, it includes promoter, herbicide tolerance genes and polyadenylic acid Change (polyadenylic acid (polyA)) terminator sequence.

Figure 73 depicts the plasmid pDAB7422 built as described in embodiment 18E.The plasmid includes the matter of on position 1 Complete promoter-transcript unit (PTU) in grain main chain (backbone), it includes promoter, herbicide tolerance genes and gathers Polyadenylation (polyadenylic acid) terminator sequence.

Figure 74 depicts the plasmid pDAB7452 built as described in embodiment 18E.The plasmid includes the matter of on position 2 Complete promoter-transcript unit (PTU) in grain main chain, it is (poly- that it includes promoter, herbicide tolerance genes and polyadenylation Adenylic acid) terminator sequence.

Figure 75 is the schematic diagram for depicting exemplary non-autonomous herbicide tolerance genes expression cassette.The structure such as embodiment Imperfect promoter-transcript unit (PTU) is included as described in 18F, it includes herbicide tolerance genes and polyadenylation (polyadenylic acid) terminator sequence.

Figure 76 depicts the plasmid pDAB7423 built as described in embodiment 18G.The plasmid includes the matter of on position 1 Imperfect promoter-transcript unit (PTU) in grain main chain, it includes herbicide tolerance genes and polyadenylation (poly- adenosine Acid) terminator sequence.

Figure 77 depicts the plasmid pDAB7454 built as described in embodiment 18G.The plasmid is as described in embodiment 18G As comprising the imperfect promoter-transcript unit (PTU) in the plasmid backbone of on position 2, it includes herbicide tolerant base Cause and polyadenylation (polyadenylic acid) terminator sequence.

Figure 78 depicts plasmid pDAB7424 (a kind of exemplary Jing built as described in embodiment 18HThe autonomous donor in position 1 of reorganization (adapt)).

Figure 79 depicts plasmid pDAB7425 (a kind of exemplary Jing built as described in embodiment 18HThe autonomous donor in position 1 of reorganization).

Figure 80 depicts the plasmid pDAB7426 built as described in embodiment 18H.PDAB7426 is a kind of combination matter Grain, it includes the autonomous donor in position 1 and ZFN expression cassettes.

Figure 81 depicts the plasmid pDAB7427 built as described in embodiment 18H.PDAB7427 is a kind of combination matter Grain, it includes the autonomous donor in position 1 and ZFN expression cassettes.

Figure 82 depicts the amplification of the donor dna specific sequence from genomic DNA.The presence of 317bp products judges Go out to insert the presence that Semen Maydiss corpus callosum (callus) is the donor dna comprising pat gene in #61-72 genomes, such as implement Described in example 20C.HiII indicates wild type negative control.

Figure 83 depicts the amplification on 5 ' borders between the special maize genomic sequence of donor dna and IPP2K.Such as embodiment Described in 21A, according to the presence of the DNA fragmentation of 1.65Kbp judge from donor to targeted integration in IPP2K genes it is derivative again Secondary PCR primer (secondary PCR product).HiII indicates wild type negative control.

Figure 84 depicts the amplification on 3 ' borders between the special maize genomic sequence of donor dna and IPP2K.Such as embodiment Described in 21A, according to the presence of the DNA fragmentation of 1.99Kbp judge from donor to targeted integration in IPP2K genes it is derivative again Secondary PCR primer.HiII indicates wild type negative control.

Figure 85 depicts the amplification on upstream (the 5 ') border between genome and donor.As described in embodiment 21B, according to big The presence of the little DNA fragmentation for 1.35Kbp is judged derivative to targeted integration in IPP2K genes (5 ' border) from donor PCR primer.HiII indicates wild type negative control.

Figure 86 depicts the amplification on downstream (the 3 ') border between donor and genome.As described in embodiment 21B, according to big The presence of the little DNA fragmentation for 1.66Kbp is judged derivative to targeted integration in IPP2K genes (3 ' border) from donor PCR primer.HiII indicates wild type negative control.

Figure 87 depicts sequence (the SEQ ID NO of the homology flank of position 15 '：171).

Figure 88 depicts sequence (the SEQ ID NO of the homology flank of position 13 '：172).

Figure 89 depicts sequence (the SEQ ID NO of the homology flank of position 25 '：139).

Figure 90 depicts sequence (the SEQ ID NO of the homology flank of position 23 '：140).

Figure 91 depicts sequence (the SEQ IDNO of upstream (5 ' -) the IPP2K genome sequences of ZFN target areas：141).

Figure 92 depicts sequence (the SEQ IDNO of downstream (3 ' -) the IPP2K genome sequences of ZFN target areas：142).

Detailed description of the invention

Disclosed herein is comprising the zinc finger Binding peptide containing Cys-Cys-His-Cys form non-standard zinc fingers (ZFP) compositionss.Because zinc coordination provides main folding energy for zinc finger, the adjustment of zinc coordination residue provides one kind For the easy means that modification refers to stability and structure, stability and structure are produced to various important functional characteristics of zinc finger protein It is raw to affect, the interaction, DNA binding specificities and affinity and work(including such as cellular half-life and other cytokines The relative orientation in energy domain.

There is display, (such as those are in U.S. Patent application for the zinc finger protein comprising non-standard zinc finger No.20030108880；20060246567；Disclosed in 20060246588) combine DNA and change transcription.However, After being impregnated in Zinc finger nuclease (ZFN see, for example, U.S. Patent Application Publication text No.2005/0064474), these are previous The non-standard zinc finger protein of description shows sometimes not good enough (sub-optimal) activity in terms of cutting target DNA.

Described herein is the zinc finger protein comprising one or more CCHC zinc fingers, wherein C-terminal zinc coordination residue pair The particular sequence of surrounding has changed by.What is be also described herein is the fusion egg comprising these through the non-standard zinc finger of optimization In vain, such as Zinc finger nuclease (ZFN), wherein the ZFN is made with the cutting realized with ZFN of the use comprising specification (CCHH) zinc finger With suitable speed or ratio (rate) cutting target DNA.

Fused polypeptide disclosed herein can strengthen or suppressor gene transcription and/or cutting target sequence.Additionally provide The polynucleotide and coding for encoding the non-standard zinc finger through optimizing include one or more non-standard zinc fingers for passing through optimization The polynucleotide of fusion protein.Pharmaceutical composition is provided in addition, and it includes the treatment combined with pharmaceutical acceptable carrier has Any improvement of described herein any zinc finger-coding of nucleotide Binding peptide or its functional fragment or therapeutically effective amount of effect amount The nucleotide sequence of zinc finger-nucleotide Binding peptide or its functional fragment.There is also provided agriculturally useful compositions, it is included and agriculture Learn the described herein any zinc finger-nucleotide Binding peptide or its functional fragment of the agronomy effective dose of acceptable carriers combination Or any improvement zinc finger-nucleotide Binding peptide of coding or the nucleotide sequence of its functional fragment of agronomy effective dose.Also Providing can be with reference to the screening technique of the improvement zinc finger-nucleotide Binding peptide of genome sequence for acquisition.

Genome sequence is present in chromosome, episome, organelle gene group (such as mitochondrion, Ye Lv including those Body), the nucleic acid of any other type present in artificial chromosome and cell (such as extension increasing sequence, double minute chromosome and Endogenous or infection antibacterial and the genome of virus) in.Genome sequence can be normal (i.e. wild type) or mutation 's；Mutant nucleotide sequence can include such as insertion, deletion, replacement, transposition, rearrangement, and/or point mutation.Genome sequence can be with Comprising one of many different allele.

Current techique

Unless otherwise stated, the preparation of compositions disclosed herein and use and the enforcement of method adopts molecular biosciences , biochemistry, chromatin Structure and analysis, the routine techniquess calculated in chemistry, cell culture, recombinant DNA and association area, These technologies are within the skill of the art.These technologies have in the literature comprehensively explanation.See, for example, Sambrook Deng MOLECULAR CLONING：ALABORATORY MANUAL, the second edition, Cold Spring Harbor Laboratory Press, 1989 and the third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley ＆ Sons, New York, 1987 and regularly update；Book series METHODSIN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATINSTRUCTURE AND FUNCTION, the third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, volume 304, " Chromatin " (P.M.Wassarman and A.P.Wolffe Compile), Academic Press, San Diego, 1999；And METHODS IN MOLECULARBIOLOGY, volume 119, " Chromatin Protocols " (P.B.Becker volumes), Humana Press, Totowa, 1999.

Definition

Term " nucleic acid ", " polynucleotide " and " oligonucleotide " is used interchangeably, and refers in linear or cyclic conformation, And or single-stranded or double chain form deoxyribonucleotide or ribonucleotide polymer.For purposes of this disclosure, this A little terms are not interpreted as the restriction with regard to polymer length.The term can cover the known analog and alkali of natural nucleotide There is the nucleotide (such as phosphorothioate backbone) of modification in base, sugar and/or phosphoric acid module.In general, specific nucleotide Analog has identical base pairing specificity；I.e. the analog of A can carry out base pairing with T.

Term " polypeptide ", " peptide " and " protein " is used interchangeably, and refers to the polymer of amino acid residue.The term is also applied It is that the chemical analog of corresponding naturally occurring aminoacid or the aminoacid of modified derivative gather in wherein one or more aminoacid Compound.

" with reference to " refers to interaction sequence-specific, non-covalent (such as between protein and nucleic acid) between macromole.Knot Close all the components for interacting not all necessarily sequence-specific (such as contacting with phosphate moiety in DNA backbone), only Sequence-specific as overall interaction.Usually, such interaction is characterised by dissociation constant (K_d) For 10^-6M^-1Or it is lower." affinity " refers to bond strength：Binding affinity is raised and K_dReduce related.

" associated proteins " refer to the protein of another molecule of Non-covalent binding.Associated proteins can be with reference to such as DNA point Sub (DBP), RNA molecule (rna binding protein) and/or protein molecule (protein-binding proteins).In protein In the case of associated proteins, it can with reference to itself (with formed homodimer, homotrimer, etc.) and/or it can tie Close the one or more different protein of one or more molecules.Associated proteins can have lives more than a type of combination Property.For example, there is zinc finger protein DNA to combine, RNA is combined and protein binding activity.

" zinc-finger DNA Binding Protein " (or binding structural domain) refers to via one or more zinc fingers, with sequence-specific fashion With reference to the protein of DNA or compared with the domain in larger protein, the zinc finger is the amino acid sequence region in binding structural domain And its structure by zinc ion coordination stabilisation.Term zinc-finger DNA Binding Protein is commonly abbreviated as zinc finger protein or ZFP.

Zinc finger binding domain can be with " transformation or through engineering approaches " (engineer) into reference to predetermined nucleotide sequence.Design With the non-limitative example that selection is the method for through engineering approaches zinc finger protein.The zinc finger protein of design is not present in nature Protein, its design/composition be derived mainly from reasonable standard (rationalcriteria).Reasonable standard for design includes Using alternative rule and computerized Algorithm, in the data base for the existing ZFP designs of process storage and with reference to data message Information.See, for example, United States Patent (USP) 6,140,081；6,453,242；6,534,261；And 6,785,613；Referring also to WO 98/ 53058；WO98/53059；WO 98/53060；WO 02/016536 and WO 03/016496；And United States Patent (USP) 6,746,838； 6,866,997；And 7,030,215.

" selection " zinc finger protein refers to the protein not found in nature, and its generation is derived mainly from empirical method, all As phage display, interaction trap or heterocomplex are selected.See, for example, US5,789,538；US 5,925,523；US 6, 007,988；US 6,013,453；US 6,200,759；US6,733,970；US RE39,229；And WO 95/19431；WO 96/06166；WO 98/53057；WO 98/54311；WO 00/27878；WO 01/60970；WO 01/88197 and WO 02/ 099084。

" unconventional " zinc finger protein refers to the protein comprising non-standard (non-C2H2) zinc finger.Thus, with it is naturally occurring C2H2 zinc finger proteins are compared, replacement of the non-standard zinc finger comprising at least one aminoacid, addition and/or deletion.Non-standard zinc finger Non-limitative example include that those are coordinated residue comprising Cys-Cys-His-Cys (such as C3H) (from amino to carboxyl) zinc.

" homologous sequence " refers to the sequence iden with the second sequences share to a certain degree, and its sequence can be with the second sequence The sequence identical First ray of row." homologous but differ sequence " refers to that the sequence with the second sequences share to a certain degree is same Property, but the First ray that its sequence is differed with the sequence of the second sequence.For example, wild-type sequence comprising mutated genes The sequence homology of polynucleotide and mutated genes but differ.In certain embodiments, the homology between two sequences Degree be enough to allow to carry out homologous recombination using normal cell mechanism between them.Two sequences that are homologous but differing can be Any length, and their non-homology degree can be as small as single nucleotide acid (for example for by targeting homologous recombination come Suppressor group point mutation) or greatly to 10 kilobase or more (such as insertion gene at the predetermined site in chromosome). There need not be equal length comprising two polynucleotide that are homologous but differing sequence.It is, for example possible to use 20 and 10,000 Exogenous polynucleotide (i.e. donor polynucleotide) between nucleotide or nucleotide pair.

Technology for determining nucleic acid and amino acid sequence identity is known in the art.Typically, such technology bag Include the nucleotide sequence to genetic testing mRNA and/or determine aminoacid sequence by coded by it, and by these sequences and the Dinucleotide or aminoacid sequence are compared.Genome sequence can also by this way be measured and compare.It is general and Speech, homogeneity refers to that accurate nucleotide is right with nucleotide (nucleotide-to-nucleotide) between two polynucleotide sequences Should or two peptide sequences between accurately aminoacid it is corresponding with aminoacid (amino acid-to-amino acid).Two or more Multiple sequences (polynucleotide or aminoacid) can be compared by determining their percentage identities.Two sequences The percentage identities of (either nucleotide sequence or aminoacid sequence) are the numbers of the accurate match between two aligned sequences Mesh divided by shorter sequence length and be multiplied by 100.The approximation ratio of nucleotide sequence is to by Smith and Waterman, Advances in Applied Mathematics 2：The local homology algorithm of 482-489 (1981) is provided.The algorithm can be by using such as Lower rating matrix and be applied to aminoacid sequence, the rating matrix is by Dayhoff, Atlas of Protein Sequences And Structure, M.O.Dayhoff are compiled, 5suppl.3：353-358, National Biomedical Research Foundation, Washington, D.C., USA are developed, and by Gribskov, Nucl.Acids Res.14 (6)：6745- 6763 (1986) standardization.The algorithm determines the exemplary execution of percent sequence homogeneity by Genetics Computer Group (Madison, WI) is provided in " best fit " (" BestFit ") utility application (utility application). The default parameter of this method is recorded in Wisconsin Sequence Analysis Package ProgramManual, and the 8th Version (1995) (being available from Genetics Computer Group, Madison, WI).Percentage ratio is set up in the background of the disclosure The exemplary methods of homogeneity are to use MPSRCH program bags, and the copyright of the MPSRCH program bags is University of Edinburgh owns, and is developed by John F.Collins and Shane S.Sturrok, and by IntelliGenetics, Inc. (Mountain View, CA) sells.From this set program bag, Smith-Waterman algorithms can be adopted, wherein by default parameter For grade form, (it is 12 that for example breach opens point penalty, and gap extension penalty is 1, and breach is for 6).From produced data, " With " value reflection sequence iden.Other programs for being suitable to percentage identities or similarity between the sequence of calculation are that this area is universal Know, for example, another kind of alignment programs are the BLAST being used together with default parameter.It is, for example possible to use BLASTN and BLASTP, it uses following default parameter：Genetic code=standard；Filter (filter)=nothing；Chain=double-strand (both)；Retention (cutoff)=60；Expect (expect)=10；Matrix (Matrix)=BLOSUM62；Description (Descriptions)=50 Sequence；Sequence (sort by)=HIGH SCORE；Data base (Databases)=nonredundant, GenBank+EMBL+DDBJ+ PDB+GenBank CDS translate+Swiss protein+Spupdate+PIR.The details of these programs can be looked on the internet Arrive.With regard to sequence described herein, the expected degree scope of sequence iden is about 35% to 100% and any integer therebetween Value.Typically, percentage identities are at least 35%-40% between sequence；40%-45%；45%-50%；50%-60%； 60%-70%；70-75%, more preferably preferred 80-82%, 85%-90%, even more preferably 92%, even more preferably from 95%, and And most preferably 98% sequence iden.

Or, the degree of sequence similarity can be determined as follows between polynucleotide, that is, allowing that homologous interval formation is stable Multi-nucleotide hybrid is carried out under conditions of duplex, single-stranded specific nucleic acid enzyme is then used by and is digested, and determined after digestion The size of fragment.If according to the measure using said method, two nucleotide sequences or two peptide sequences limit length point Represent at least about 70%-75%, more preferably preferred 80%-82%, 85%-90% in son, even more preferably 92%, it is also more excellent 95%, and most preferably 98% sequence iden is selected, then described two nucleotide sequences or two peptide sequences are mutually substantially It is homologous.As used herein, it is substantially homologous also to refer to the sequence that complete homogeneity is shown relative to specified DNA or peptide sequence Row.Substantially homologous DNA sequence can in Southern hybrid experiments in such as stringent condition (by that particular system institute It is determined that) under identify.Determine that Suitable hybridization conditions are within the skill of the art.Sambrook etc. is see, for example, is seen above； Nucleic Acid Hybridization：APractical Approach, edit B.D.Hames and SJ.Higgins, (1985)Oxford；Washington, DC；IRL Press.

The selective cross of two nucleic acid fragments can be determined as follows.The degree of sequence iden between two nucleic acid molecules Affect the efficiency and intensity of such intermolecular hybridisation events.To I haven't seen you for ages, part suppresses identical sequence to part identical nucleotide sequence The hybridization of row and target molecule.Suppression to the hybridization of identical sequence can be commented using hybridisation assays well known in the art Estimate (such as Southern (DNA) trace, Northern (RNA) trace, solution hybridization etc., referring to Sambrook etc., Molecular Cloning：A LaboratoryManual, the second edition, (1989) Cold Spring Harbor, N.Y.).This Class algoscopy can come real using different degrees of selectivity (such as using the different condition from low stringency to high stringency) Apply.If using low stringency, then the shortage of non-specific binding can be assessed as follows, i.e., using even shortage part Second probe (for example there is the probe less than about 30% sequence iden with target molecule) of degree of sequence homogeneity so that During without non-specific binding events, second probe will not hybridize with target.

When using based on the detecting system for hybridizing, select and the nucleic probe with reference to nucleic acid array complementation, then pass through Select suitable condition so that the probe and canonical sequence selective cross or are combined forming duplex molecule each other.Energy Enough nucleic acid molecules of selective cross canonical sequence under the conditions of moderate stringency hybridization typically hybridize in following condition, institute The condition of stating allows that detection has at least about 70% sequence iden, length at least about with selected nucleic acid probe sequence The target nucleic acid sequence of 10-14 nucleotide.Stringent hybridization condition typically allows to detect have greatly with selected nucleic acid probe sequence In about 90-95% sequence idens, the target nucleic acid sequence that length is at least about 10-14 nucleotide.For probe/ginseng Can be such as this according to the useful hybridization conditions of sequence hybridization (its middle probe and canonical sequence have the sequence iden of specific degrees) Determine as field is known and (see, for example, Nucleic Acid Hybridization：APractical Approach, compile Collect B.D.Hames and S.J.Higgins, (1985) Oxford；Washington, DC；IRL Press).

Hybridization conditions are well known to the skilled person.Hybridization stringency refer to hybridization conditions be unfavorable for being formed containing The heterocomplex institute degree to which of mismatched nucleotide, wherein higher stringency and the toleration phase relatively low to mispairing heterocomplex Close.Affect Hybridization stringency factor be well known to the skilled person, and including but not limited to temperature, pH, from Sub- intensity and organic solvent (such as Methanamide and dimethyl sulfoxide) concentration.As one of skill in the art will recognize, hybridize Stringency increases because temperature rising, ionic strength reduction and solvent strength are reduced.

With regard to the stringency for hybridizing, it is well known in the art that can set up specific tight using many conditions of equivalent Lattice, this is realized by changing such as following factors：The length and property of sequence, the base composition of various sequences, salt and In concentration, the hybridization solution of other hybridization solution components with the presence or absence of sealer (such as dextran sulfate and Polyethylene Glycol), Hybridization temperature and time parameter；And change wash conditions realizing.The selection of the concrete set of hybridization conditions follows this The standard method in field carries out (see, for example, Sambrook etc., Molecular Cloning：A Laboratory Manual, The second edition, (1989) Cold Spring Harbor, N.Y.).

" restructuring " refers to the process of crossing over inheritance information between two polynucleotide.For purposes of this disclosure, " homologous recombination (HR) the specialized form of the such exchange occurred in the repair process of such as cell double center chain fracture " is referred to.This process will Nucleotide sequence homology is sought, the mould of " target " molecule (experienced the molecule of double-strand break) is carried out using " donor " molecule Plate is repaired, and because it causes hereditary information to be transferred to target from donor, thus it is referred to variously as " non-crossing type gene turn Change " (" non-crossovergene conversion ") or " short-track transcription frequency " (" short tract gene conversion”).On the premise of being not intended to be limited by any particular theory, this transfer can involve fracture target and The mispairing correction of the heteroduplex DNA formed between donor, and/or " synthesis dependency chain annealing " (" synthesis- Dependent strand annealing ") (wherein carrying out resynthesis using donor can be changed into the hereditary information of a target part), And/or correlated process.The HR of this specialization typically results in the change of target molecular sequences so that donor polynucleotide sequence In part or whole incorporation target polynucleotide.

" cutting " refers to the fracture of DNA molecular covalent backbone.Cutting can be started by various methods, including but not limited to The enzymatic or chemical hydrolysis of phosphodiester bond.Single-stranded cutting and double-strand are cut both possible, and double-strand cutting can be with Occur because of two different single-stranded cutting events.DNA cuttings can cause the generation of flat end or staggered end.At some In embodiment, by fused polypeptide for the cutting of targeting double-stranded DNA.

" cutting domain " includes one or more peptide sequence for possessing the catalysis activity to DNA cuttings.Cutting structure Domain may be embodied in wall scroll polypeptide chain, or cleavage activity can be derived from the combination of two (or more) polypeptides.

" cutting half domain " refer to from the second polypeptide (or identical or different) combination and formed and lived with cutting The peptide sequence of the complex of property (preferred double-strand cleavage activity).

Term " cutting domain " and " cutting half domain " include the wild type of cutting domain or cutting half domain Domain and part or mutant, it retains multimerization (such as dimerization) to form the ability of feature cutting domain.

" chromatin " refers to the nucleoprotein structure comprising cellular genome.Cyto-chromatin comprising nucleic acid (mainly DNA) and Protein (including histone and NHC protein).Most of eukaryotic cell chromatins are deposited in nucleosome form , wherein DNA of the nucleosome core comprising about 150 base pairs, the DNA be respectively histone H2A, H2B, H3 comprising double Combine with eight aggressiveness of H4；And bonding pad DNA (being variable-length, depending on organism) prolongs between nucleosome core Stretch.Usually, a molecule histone h1 is combined with bonding pad DNA.For purposes of this disclosure, term " chromatin " is intended to cover Both all types of Nuclear extract, procaryotic and Eukaryotic.Cyto-chromatin includes chromosomal pattern and attached Both plus the chromatin of build.

" chromosome " refers to the chromatin complex all or in part comprising cellular genome.Generally, cellular genome Its caryogram is characterised by, it is the set (collection) of all chromosomes for constituting cellular genome.Cellular genome can Comprising one or more chromosomes.

" episome " (episome) refers to replication form nucleic acid, nucleoprotein complex or comprising not being cell chromosome caryogram one The other structures of partial nucleic acid.Episomal example includes plasmid and some viral genome.

" can and area " (accessible region) refers to the following site in cyto-chromatin, present in its amplifying nucleic acid Target site can be identified the exogenous molecules of the target site and combine.On the premise of being not intended to be limited by any particular theory, recognize For can and area be the region that is not packed in nucleosomal structure.It is different can and plot structure generally can by its to chemistry and The sensitivity of enzyme probe (such as nuclease) is detecting.

" target site " or " target sequence " refers to can be with reference to (if exist to be enough to make with reference to the condition for occurring to binding molecule Words) the nucleotide sequence that is defined of nucleic acid moiety.For example ,-the GAATTC-3 ' of sequence 5 ' is Eco RI restriction endonucleases Target site.

" external source " molecule refer to be not present under normal circumstances in cell but can pass through one or more heredity, it is biochemical Or other methods and import the molecule in cell." in normally present in cell " be relative to cell the specific stage of development and Environmental condition and determine.Thus, for example, it is relative to adult muscle to exist only in the molecule in the embryo development procedure of muscle The exogenous molecules of cell.Similarly, the molecule for being induced by heat shock is the exogenous molecules relative to non-heat-shocked cell.External source Molecule can include function (functioning) pattern or just of (malfunctioning) endogenous molecule of such as disfunction The disfunction pattern of the endogenous molecule of normal function.

Exogenous molecules can small molecule (such as being generated by combined chemical method) or macromole (such as egg White matter, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide), any modified derivative or any of above-mentioned molecule Complex comprising one or more above-mentioned molecule etc..Nucleic acid includes DNA and RNA, can be single-stranded or double-stranded；It can be line Property, branch or ring-type；And can be any length.Nucleic acid includes that those can form the nucleic acid of duplex, with And the nucleic acid of formation triplex.See, for example, United States Patent (USP) No.5,176,996 and 5,422,251.Protein is included but is not limited to DBP, transcription factor, Chromatin remodeling factors (chromatin remodeling factor), methylate DNA knot It is hop protein, polymerase, methylase, demethylase, acetylase (acetylase), deacetylase, kinases, phosphatase, whole Synthase, recombinase, ligase, topoisomerase, gyrase and unwindase.

Exogenous molecules can be the molecule with endogenous molecule same type, such as exogenous proteins or nucleic acid.For example, external source Nucleic acid can be thin comprising infectious virus genome, Agrobacterium tumefaciens (Agrogacteriumtumefacians) T chains, importing Plasmid or episome in born of the same parents or the chromosome being not present under normal circumstances in cell.But, exogenous nucleic acid or polynucleotide Or identical sequence homologous with endogenous sequence can be included.Relative to specific endogenous gene group region, " exogenous array " refers to and do not exist In the nucleotide sequence in the region.The exogenous array may be present in another endogenous chromosome position or its and may be not present at all In genome.Thus, exogenous polynucleotide can include both external source and endogenous sequence：For example, flank is same with genome area The transgenic of the sequence in source.It is described below such exogenous nucleic acid used in the method recombinated for targeted integration and targeting.With In be known to those skilled in the art the method in exogenous molecules importing cell, and including but not limited to lipid is situated between The transfer (i.e. liposome, it includes neutral lipid and cation lipid) led, electroporation, direct injection, cell fusion, particle Hong Hit, coprecipitation of calcium phosphate, DEAE- dextrans mediation transfer and viral vector mediation transfer.

Comparatively, " endogenous " molecule refers to just to be normally present under certain environmental conditions is in the specific of specific stage of development Molecule in cell.For example, endogenous nucleic acid can include chromosome, the genome of mitochondrion, chloroplast or other organelles, or Naturally occurring additional build nucleic acid.Other endogenous molecule can include protein, such as transcription factor and enzyme.

" fusion (thing) " molecule refer to wherein have two or more subunit molecules connections (for example covalently) together point Son.Subunit molecules can be the molecule of identical chemical type, or can be the molecule of different chemical types.First type The example of fusion molecule includes but is not limited to fusion protein (such as fusion between ZFPDNA binding structural domains and cutting domain Thing) and integrative nucleic acid (for example encoding the nucleic acid of fusion protein mentioned above).The example of the fusion molecule of second type includes But it is not limited to form the fusions between the nucleic acid of triplex and polypeptide, and the fusions between minor groove binders and nucleic acid.

Expression of the fusion protein in cell can be derived from and for the fusion protein be delivered to cell or by the way that coding is somebody's turn to do The polynucleotide of fusion protein are delivered to cell, wherein the polynucleotide are transcribed, and transcript is translated, to generate State fusion protein.Expression of the protein in cell can also involve trans-splicing, polypeptide cutting and polypeptide connection.For will be many Nucleotide and polypeptide are delivered to the method for cell and propose in the other places of the disclosure.

" gene " includes for purposes of this disclosure the region of DNA domain (seeing below) of encoding gene product (, and regulator gene All region of DNA domains that product is generated, no matter whether such regulatory sequence is adjacent to coded sequence and/or transcribed sequence.Thus, base Cause including but not necessarily limited to promoter sequence, terminator, translational regulation sequence (such as ribosome binding site and internal ribosome Entry site), enhancer, silent gene (silencer), insulator (insulator), boundary element, replication orgin, substrate Attachment site and locus control region.

" gene expression " refers to that information contained is converted into gene outcome in gene.Gene outcome can be the direct transcription of gene Product (such as mRNA, tRNA, rRNA, antisense RNA, ribozyme, the RNA of structural RNA or any other types) or by mRNA The generated protein of translation.Gene outcome is also included by capping, polyadenylation, methylate and the process such as edit and modified RNA, and for example, by methylating, acetylation, phosphorylation, ubiquitination, ADP- ribosylation, myristyl and glycosyl Change modified protein.

" regulation and control " of gene expression refer to the change of gene activity.Expression regulation can include but is not limited to gene activation and base Because preventing.

" plant " cell includes but is not limited to the cell of unifacial leaf (monocot) or dicotyledonous (dicot) plant.Unifacial leaf The non-limitative example of plant includes cereal, such as Semen Maydiss, rice, Fructus Hordei Vulgaris, Herba bromi japonici, Semen Tritici aestivi, Sorghum vulgare Pers., rye (Secale cereale L.), Caulis Sacchari sinensis, spinach Trailing plants, Bulbus Allii Cepae, Fructus Musae and Cortex cocois radiciss.The non-limitative example of dicotyledon includes Nicotiana tabacum L., Fructus Lycopersici esculenti, Helianthi, cotton, Radix Betae, Ma Ling Potato, Caulis et Folium Lactucae sativae, Fructus Melo (melon), Semen sojae atricolor, Semen Brassicae Campestriss (Brassica campestris L) and Herba Medicaginiss.Plant cell can come from plant any part and/ Or from any stage of development of plants.

" area-of-interest " refers to any cyto-chromatin region for expecting to be combined with exogenous molecules, such as gene or base Because of interior or neighbouring with gene non-coding sequence.With reference to can be in order to targeting DNA cutting and/or targeting restructuring purpose.Example Such as, area-of-interest may reside in chromosome, episome, organelle gene group (such as mitochondrial, chloroplast) or sense In metachromia viral genome.Area-of-interest can be in the coding region of gene, noncoding region (the such as leading sequence of transcription Row, tailer sequence or intron) in or nontranscribed domain (or upstream of coding region or coding region downstream) in.Area-of-interest Length can be as small as single nucleotide acid pair or up to 25,000 nucleotide pair, or any integer-valued nucleotide pair.

Term " being operatively connected " with regard to two or more components (such as sequential element) side by side when be used interchangeably, its In arrangement is carried out to the component so that two components are all worked orderly, and allow in the component that at least one can be situated between Lead the probability for putting at least one function in other components.For example, if transcriptional regulatory sequences response it is a kind of or The transcriptional level of coded sequence is controlled during various transcription regulaton factor presence or absence, then the transcriptional regulatory sequences (such as promoter) It is operatively connected to the coded sequence.Transcriptional regulatory sequences are typically operatively connected with coded sequence cisly, but are not required to directly It is adjacent.For example, enhancer is the transcriptional regulatory sequences being operatively connected with coded sequence, even if they are not continuous.

With regard to fused polypeptide, term " being operatively connected " can refer to the work(that each component is performed when another component is connected Can be with its function identical truth that it can be performed when not so being connected.For example, melt with regard to wherein ZFP DNA binding structural domains The fused polypeptide of cutting domain is bonded to, if in the fused polypeptide, ZFP DNA binding structural domains part can be with reference to its target Site and/or its binding site, and cutting domain can cut the DNA near target site, then the ZFP DNA integrated structures Domain and the cutting domain are operatively connected.

" functional fragment " of protein, polypeptide or nucleic acid refers to that its sequence is different from full length protein, polypeptide or nucleic acid, but Still retain and full length protein, the protein of polypeptide or nucleic acid identical function, polypeptide or nucleic acid.Functional fragment can possess with Corresponding natural molecule compares more, less or identical number of residues, and/or can include at one or many places aminoacid or nucleoside Acid is substituted.Method for determining nucleic acid function (such as the ability of encoding function and another nucleic acid hybridization) is known in this field 's.Similarly, the method for determining protein function is known.For example, polypeptide combine the function of DNA can be for example, by Filter is determined with reference to (filter-binding), electrophoresis mobility shift or immunoprecipitation assay.Cutting to DNA can To be determined by gel electrophoresiss.Referring to Ausubel etc., see above.A kind of protein and another kind of protein interaction Ability can be determined for example, by co-immunoprecipitation, two-hybrid assay or complementation (both heredity and biochemistry).Referring to Such as Fields etc., (1989) Nature340：245-246；United States Patent (USP) No.5,585,245 and PCT WO 98/44350.

Zinc finger binding domain

Described herein is many nucleoside in non-standard Zinc finger binding domain and these Zinc finger binding domains of coding Acid.In certain embodiments, non-standard Zinc finger binding domain described herein is C3H zinc fingers, and the conservative zinc of two of which is matched somebody with somebody One of hyte histidine residue is converted into cysteine.In other enforcement embodiment, the histidine residues of most C-terminal are changed For cysteine residues, generate " CCHC albumen ".

Zinc finger binding domain can include one or more zinc fingers (such as 2,3,4,5,6,7,8,9 or more zinc fingers), And can be transformed into reference to any target sequence (such as genome sequence).Zinc finger binding domain can with reference to DNA, RNA and/or Protein.Typically, the length in single zincfinger motif domain is for about 30 aminoacid.Zinc finger includes specification C₂H₂Zinc finger (i.e. those its Middle zinc ion is by two cysteine and the zinc finger of two histidine residues coordination) and non-standard zinc finger (including such as C3H zinc Both refer to, i.e., those wherein zinc ioies are by three cysteine residues and the zinc finger of a histidine residues coordination).Referring also to U.S. Patent application No.20030108880；20060246567；With 20060246588, their disclosure is included by referring to Content.

Structural research has confirmed that specification Zinc finger domain (motif) (is maintained at constant comprising two comprising two β lamellas Cysteine residues β-bend in) and α spiral (including two constant histidine residues), they pass through two and half Cystine and two histidine coordinated Zn atoms and with the holding of specific conformation.Non-standard zinc finger disclosed herein keeps this Plant β-β-α structures.

Non-standard zinc finger described herein can be naturally occurring Zinc finger binding domain.More typically, however, it is described herein Non-standard zinc finger includes one or more following zinc finger components, wherein with one or more zinc of amino acid substitution at least one Coordination cysteine or histidine residues.For example, in certain embodiments, specification zinc finger binding modules are replaced with Cys residues C-terminal His residue.

CCHC zinc fingers described herein can also include amino acid residue sequence in except zinc be coordinated residue in addition to one at or many places change Become (relative to naturally occurring C2H2 zinc fingers sequence).Such change can be comprising replacement, deletion, and/or insertion.Can appoint in zinc finger Where side changes aminoacid.The non-limitative example of change includes：(1) the single residue around zinc coordination residue being changed is replaced Generation；(2) the additional residue insertion before or after the zinc coordination residue being changed, (such as the His residues in most C-terminal are changed In the case of Cys, the addition of additional amino acid residue can promote zinc to be coordinated by compensating shorter cysteine side chain)； And/or (3) replace the residue positioned between His the and Cys residues of naturally occurring CCHC zinc fingers to the phase of non-standard CCHC zinc fingers In answering region.

In certain embodiments, zinc finger protein described herein includes at least one zinc finger, and it includes that non-standard is (non- C2H2 the zinc co-ordination area that) zinc finger, wherein non-standard zinc finger have the spiral part and wherein spiral part that involve DNA combinations is included Aminoacid sequence HX₁X₂RCX_L(SEQ ID NO：2)；And wherein zinc finger protein is transformed into and combines target sequence.In some embodiment party In case, X₁It is A or K or T；X₂It is Q or E or R；And X_LIt is G.

In other embodiments, non-standard zinc finger described herein has general structure：Cys-(X^A)_2-4-Cys-(X^B)₁₂- His-(X^C)_3-5-Cys-(X^D)_1-10(SEQ ID NO：3), wherein X^A、X^B、X^CAnd X^DRepresent any aminoacid.In X^CIt is residual comprising 3 In the embodiment of base, (i) compared with specification CCHC zinc finger, change at least one in these residues；And/or (ii) X^DComprising with Specification CCHH zinc finger compare at least one at delete, substitute or insert.In certain embodiments, X^DComprising sequence QLV or QKP.In other embodiments, X^DComprising one or more (such as 1,2,3,4,5,6,7,8,9 It is individual or 10) Gly (G) residue.

Show the partial amino-acid series of exemplary non-standard zinc finger (in the 3rd zinc in table 1, table 2, table 3 and table 4 The C-terminal of coordination residue is simultaneously coordinated residue including the 3rd zinc).In all of table, two most C-terminal (i.e. the 3rd and the 4th) zinc Coordination residue (H and C) is underlined.Shown with double underline and refer to sequence (the 2nd of table 1 and table 3 with " wild type " non-standard The change (for example substitute, insert, deleting) OK) compared.

Table 1

Table 2

Table 3

Table 4

As described above, ZFP may include any number of Zinc finger binding domain, for example, at least 3 zinc fingers.Additionally, one It is individual, more than one or all zinc fingers can be non-standard zinc finger described herein.

In certain embodiments, the most C-terminal of the zinc finger protein of multiple fingers refers to comprising specification zinc finger.In other embodiments In, the most C-terminal of the zinc finger protein of multiple fingers refers to and refers to comprising CCHC described herein, for example, be coordinated Cys residue C-terminals comprising most C-terminal zinc One at or the CCHC of many places aminoacid insertion refer to.Referring to embodiment 1-5, the zinc finger protein of 4 fingers, its middle finger 2 are which depict (F2) and/or refer to that 4 (F4) are non-standard zinc fingers described herein.

Zinc finger binding domain can be transformed into reference to selected sequence.See, for example, Beerli etc. (2002) Nature Biotechnol.20：135-141；Pabo etc. (2001) Ann.Rev.Biochem.70：313-340；Isalan etc. (2001) Nature Biotechnol.19：656-660；Segal etc. (2001) Curr.Opin.Biotechnol.12：632-637； Choo etc. (2000) Curr.Opin.Struct.Biol.10：411-416.Compared with naturally occurring zinc finger protein, through engineering approaches Zinc finger binding domain can have new binding specificity.Remodeling method includes but is not limited to appropriate design and various types of Select (method for for example screening various different zinc finger sequences for single target nucleotide sequences).Appropriate design includes for example using Data base comprising triplet (or tetrad) nucleotide sequence and various zinc finger aminoacid sequences, wherein every kind of triplet or four Conjuncted nucleotide sequence is associated with one or more aminoacid sequence with reference to specific triplet or the zinc finger of tetrad sequence.Ginseng See for example total United States Patent (USP) 6,453,242 and 6,534,261.Other method for designing is disclosed in such as United States Patent (USP) 6,746, 838；6,785,613；6,866,997；And 7,030,215.The enhancing of Zinc finger binding domain binding specificity has been recorded in Such as total United States Patent (USP) No.6,794,136.

Exemplary system of selection (including phage display and two-hybrid system) is disclosed in United States Patent (USP) 5,789,538； 5,925,523；6,007,988；6,013,453；6,410,248；6,140,466；6,200,759；And 6,242,568；And WO 98/37186；WO 98/53057；WO 00/27878；WO 01/88197 and GB 2,338,237.

Because various zinc fingers combine (i.e. triplet) sequence of 3 nucleotide, (or the sequence of 4 nucleotide, it can be with With the overlap that the binding site of 4 nucleotide of adjacent zinc finger has a nucleotide), so tying after the transformation of Zinc finger binding domain Sequence (such as target sequence) length of conjunction will determine the number of zinc finger in through engineering approaches Zinc finger binding domain.For example, for wherein Zinc-finger motif does not combine the ZFP of overlapping sublocus, and the target sequence of 6 nucleotide is combined by the binding structural domain of 2 fingers；9 The target sequence of nucleotide is combined by the binding structural domain of 3 fingers；Etc..The individual binding site of zinc finger is (i.e. sub- in target site Site) need not to be continuous, and can be to be separated by one or several nucleotide, this depends on zinc finger in many finger binding domains Between aminoacid sequence (referring to a joint) length and property.See, for example, United States Patent (USP) 6,479,626；6,903,185 and 7, 153,949 and U.S. Patent Application Publication text No.2003/0119023；Disclosures of which is included by referring to.

In Zinc finger binding domain is referred to, adjacent zinc finger can be by the amino acid linker sequence of about 5 aminoacid more (so-called " specification " refers to a joint) or separated by one or more non-standard joints.See, for example, total United States Patent (USP) No.6,453,242 and 6,534,261.For comprise more than three finger through engineering approaches Zinc finger binding domains, some zinc fingers it Between insert longer (" unconventional ") refer to a joint sequence can improve binding structural domain combination affinity and/or specificity. See, for example, United States Patent (USP) No.6,479,626 and U.S. Patent Application Publication text No.2003/0119023, by referring to and Include disclosures of which.Thus, refer to that Zinc finger binding domain can also refer to presence and the position side of a joint in non-standard more Face is characterizing.The use of joint between longer finger may additionally facilitate combination of the zinc finger protein to the target site comprising discontinuous nucleotide. Therefore, one or more sublocus in the target site in Zinc finger binding domain can by 1,2,3,4,5 or more nucleotide that This separates.An example is only provided, the binding structural domain of 4 fingers can be with reference to the target site of 13 nucleotide, and it sequentially includes two The sublocus of individual continuous 3 nucleotide, one interleave nucleotide and two continuous triplet sublocus.

Target sublocus is the nucleotide sequence (usually 3 or 4 nucleotide) that single zinc finger is combined.However, target position Point needs not to be the multiple of three nucleotide.For example, occur intersect chain interact in the case of (see, for example, United States Patent (USP) 6, 453,242 and 6,794,136), one or more zinc fingers in many finger binding domains are individual can be with reference to overlapping tetrad Asia position Point.Referring also to United States Patent (USP) 6,746,838 and 6,866,997.An example is only provided, the binding structural domain of 3 fingers can be tied The target site of 10 nucleotide is closed, it includes the sublocus of three overlapping 4 nucleotide.

Can realize according to the method disclosed in for example total United States Patent (USP) No.6,453,242 (on September 17th, 2002) The selection of Zinc finger domain binding sequence (such as target site) in cyto-chromatin, the patent is further disclosed and combines choosing for design The method of the ZFP of sequencing row.It will be obvious to those reasonably skilled in the art that can also be selected by simple visual inspection nucleotide sequence Select target site.Thus, any means for target position point selection used in methods described herein.

Refer to that zinc finger protein can be by connection for example by designing or selecting the zinc finger individuality for obtaining to build more.Or, can The binding modules that joint (seeing above) will be made up of two zinc fingers between operating specification or longer, unconventional finger (module) it is connected to each other, is referred to and 6 protein for referring to generating 4.The module of such two fingers can be for example, by referring to more Select the finger of two target sequences adjacent, with reference to specific 6 nucleotide in the background of protein (usually 3 finger) to obtain .WO 98/53057 and U.S. Patent Application Publication text No.2003/0119023 is see, for example, by referring to it is included Disclosure.Or, the module of two fingers can be built by the individual assembling of zinc finger.

Thus, can be used alone Zinc finger domain described herein or with it is various be applied in combination Zinc finger domain described herein with Structure can be with reference to many finger zinc finger proteins of any target site.

Nucleotide or the number of nucleotide pair that distance between sequence (such as target site) is inserted between two sequences between referring to, As measured by from immediate sequence edge each other.

In the embodiment using ZFN, for example wherein dissection depends on two Zinc finger domains/cutting half structure Combination of the domain fusion molecule to separate target site, two target sites can be on the DNA of opposition.In other embodiments, Two target sites are on identical DNA.WO2005/084190 is see, for example, disclosure of which is included by referring to.

The polynucleotide of coding zinc finger or zinc finger protein are also within the scope of the present disclosure.These polynucleotide can be using mark Quasi- technology can be inserted in carrier building, and (can see below with regard to carrier and for will in the vectors into cells Polynucleotide import other disclosure of the method in cell) so that coded protein is expressed in cell.

Fusion protein

Additionally provide the fusion protein comprising one or more non-standard zinc finger components described herein.

By well known to a person skilled in the art clone and biochemistry conjugation methods are building fusion molecule.Fusion molecule Comprising the ZFP containing CCHC and such as cutting domain, cutting half domain, transcriptional activation domain, trancription repression domain, The composition of chromatin reconstitution complex, insulator structure domain, arbitrary functional fragment of these domains；And/or two or Any combinations of more functional domains or its fragment.

In certain embodiments, fusion molecule is (such as exhausted comprising improvement plant zinc finger protein and at least two functional domains Edge domain or methyl binding proteins domain, and additionally, transcriptional activation or repression domain).

Optionally, fusion molecule also includes nuclear localization signal (such as from SV40T antigens or Semen Maydiss opaque-2 (Opaque-2) NLS) and epitope tag (such as FLAG or hemagglutinin).Design fusion protein (and encode their core Acid) so that keeping translation reading frame between each component of fusions.

For design and construction of fusion protein (and encoding their polynucleotide) method be those skilled in the art Know.For example, the method for designing and building the fusion protein comprising zinc finger protein (and encoding their polynucleotide) is remembered It is loaded in total United States Patent (USP) 6,453,242 and 6,534,261.

The polynucleotide of encoding such fusion albumen are also within the scope of the present disclosure.These polynucleotide can use standard Technology can be inserted in carrier building, and (will can see below with regard to carrier and for will be many in the vectors into cells Nucleotide imports other disclosure of the method in cell).

For it is merging with ZFP DNA binding structural domains, to be used for suppressor gene expression exemplary functional domain be from The KRAB repression domains of people's KOX-1 albumen (see, for example, Thiesen etc., NewBiologist 2,363-374 (1990)； Margolin etc., Proc.Natl.Acad.Sci.USA 91,4509-4513 (1994)；Pengue etc., Nucl.Acids Res.22：2908-2914(1994)；Witzgall etc., Proc.Natl.Acad.Sci.USA 91,4514-4518 (1994)). KOX domains are also suitable as repression domain.Another kind of suitable repression domain is methyl binding structural domain albumen 2B (MBD-2B) (with regard to MBD albumen description referring also to Hendrich etc. (1999) Mamm Genome10：906-912).It is another It is protein bound with v-ErbA to plant useful repression domain.See, for example, Damm etc. (1989) Nature 339：593- 597；Evans(1989)Int.J.Cancer Suppl.4：26-28；Pain etc. (1990) New Biol.2：284-294；Sap Deng (1989) Nature340：242-244；Zenke etc. (1988) Cell 52：107-119；And (1990) Cell61 such as Zenke： 1035-1049.Other exemplary repression domain include but is not limited to Thyroid Hormone Receptors (TR), SID, MBD1, MBD2, MBD3, MBD4, MBD sample albumen, DNMT family members (such as DNMT1, DNMT3A, DNMT3B), Rb, MeCP1 and MeCP2.Ginseng See such as Zhang etc. (2000) Ann Rev Physiol 62：439-466；Bird etc. (1999) Gell 99：451-454； Tyler etc. (1999) Cell 99：443-446；Knoepfler etc. (1999) Cell 99：447-450；And Robertson etc. (2000)Nature Genet.25：338-342.Other exemplary repression domain includes but is not limited to ROM2 and AtHD2A.Ginseng See such as Chern etc. (1996) Plant Cell 8：305-321；And (2000) Plant such as Wu is J.22：19-27.

Be adapted for carrying out activate domain include HSV VP16 activation structures domain (see, for example, Hagmann etc., J.Virol.71,5952-5962 (1997))；Nuclear hormone receptor (Torchia etc. is see, for example, Curr.Opin.Cell.Biol.10：373-383(1998))；Nuclear factor kappa B p65 subunits (Bitko and Barik, J.Virol.72：5610-5618(1998)；Doyle and Hunt, Neuroreport 8：2937-2942(1997)；Liu etc., Cancer Gene Ther.5：3-28(1998))；Or artificial chimeric's functional domain, VP64 is (Seifpal etc., EMBO J.11, 4961-4968(1992))。

Other exemplary activation structure domain includes but is not limited to p300, CBP, PCAF, SRC1 PvALF and ERF-2.Referring to Such as Robyr etc. (2000) Mol.Endocrinol.14：329-347；Collingwood etc. (1999) J.Mol.Endocrinol.23：255-275；Leo etc. (2000) Gene 245：1-11；Manteuffel-Cymborowska (1999)Acta Biochim.Pol.46：77-89；McKenna etc. (1999) J.Steroid Biochem.Mol.Biol.69： 3-12；Malik etc. (2000) Trends Biochem.Sci.25：277-283；And Lemon etc. (1999) Curr.Opin.Genet.Dev.9：499-504.Other exemplary activation structure domain include but is not limited to OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7 and -8, CPRF1, CPRF4, MYC-RP/GP and TRAB1.See, for example, Ogawa etc. (2000) Gene 245：21-29；Okanami etc. (1996) Genes Cells 1：87-99；Goff etc. (1991) GenesDev.5：298- 309；Cho etc. (1999) Plant Mol.Biol.40：419-429；Ulmason etc. (1999) Proc.Natl.Acad.Sci.USA 96：5844-5849；Sprenger-Haussels etc. (2000) Plant is J.22：1-8； Gong etc. (1999) Plant Mol.Biol.41：33-44；And (1999) Proc.Natl.Acad.Sci.USA96 such as Hobo： 15,348-15,353.

Other functional domain is disclosed in for example total United States Patent (USP) No.6,933,113.Additionally, being suitable in fusion molecule Insulator structure domain, chromatin reconstitution albumen (the such as domain containing ISWI) and the methyl binding structural domain albumen for using It is recorded in the international publication WO 01/83793 and WO02/26960 for example having.

In other embodiments, fusion protein is following Zinc finger nuclease (ZFN), and it includes one or more herein The CCHC zinc fingers and cutting domain (or cutting half domain).Zinc finger can be transformed in any selected genes group region of identification Target sequence, and combination of the fusion protein to its binding site can be caused after being imported in cell, and in the base Because being cut in group region or near the genome area.The cutting can cause the genome after non-homologous end joining The nucleotide sequence in region changes (be for example mutated).Or, if many nucleoside of external source comprising the sequence homologous with genome area Acid is existed in the cell, then after the targeting cutting that ZFN is carried out, with height ratio (rate) in genome area and external source There is homologous recombination between polynucleotide.Homologous recombination can cause the targeting sequence of exogenous array to be replaced or targeted integration, and this takes Certainly in the nucleotide sequence of exogenous polynucleotide.

Non-standard zinc finger described herein provides improved cutting function after ZFN is introduced into.It is as be shown in the examples, bag The ZFN of 4 fingers referred to containing at least one CCHC described herein is carried out goodly at least as the nuclease for referring to comprising CCHH only Cutting.In certain embodiments, when C-terminal refers to comprising non-standard CCHC zinc fingers, between the 3rd and the 4th zinc coordination residue The residue of (i.e. between C-terminal His and Cys residues) is different from the residue that those are present in specification CCHH zinc finger, and in C-terminal One or more glycine residues (such as 1,2,3,4,5 or more) are inserted after Cys residues.

The cutting domain part of ZFN disclosed herein is available from any endonuclease or exonuclease.Can spread out The exemplary endonuclease of raw cutting domain includes but is not limited to restriction endonuclease and homing endonuclease.Ginseng See such as 2002-2003 catalogues, New England Biolabs, Beverly, MA；And Belfort etc. (1997) Nucleic Acids Res.25：3379-3388.The enzyme of other cutting DNA is known (such as S1 nucleases；Mung bean nuclease Enzyme；Pancreas DNA enzymatic I；Micrococcal nuclease；Yeast HO endonucleases；Referring also to (eds.) Nucleases such as Linn, Cold Spring HarborLaboratory Press, 1993).One or more in these enzymes (or its functional fragment) can be with Use as the source of cutting domain and cutting half domain.

Similarly, cutting half domain can be derived from any nuclease as set forth above or part thereof, if cutting Cutting the cleavage activity of half domain needs dimerization.If in general, fusion protein includes cutting half domain, then to base Because the targeting cutting of group DNA needs two fusion protein.Or, it is possible to use the single albumen comprising two cutting half domains Matter.Two cutting half domains can be derived from identical endonuclease, or each cutting half domain can be derived from Different endonucleases.In addition, the target site of two fusion protein be arranged with respect to one another into cause two fusion protein with The combination of each of which target site forms feature cutting domain (such as by dimerization) to allow cutting half domain Each other spatial orientation places cutting half domain.Thus, in certain embodiments, the proximal edge (near edge) of target site Separate by 5-8 nucleotide pair or by 15-18 nucleotide pair.In other embodiment, target site 10 core apart Within thuja acid pair.However, the nucleotide or nucleotide pair of any integer number being inserted between two target sites (for example from 2 to 50 nucleotide or more).In general, cut point is located between target site.

Restriction endonuclease (restriction enzyme) is present in many species, and can sequence-specific combine DNA ( At recognition site), and cutting DNA at binding site or at close binding site.Some restriction enzymes (such as IIS types) with Recognition site away from site cutting DNA, and with the combination and cutting domain that can divide.For example, IIS types enzyme Fok I are urged Change the double-strand to DNA to cut, position is recognized apart from it at 9 nucleotide of its recognition site and on another on a chain Carry out at 13 nucleotide of point.See, for example, United States Patent (USP) 5,356,802；5,436,150 and 5,487,994；And Li etc. (1992)Proc.Natl.Acad.Sci.USA 89：4275-4279；Li etc. (1993) Proc.Natl.Acad.Sci.USA 90：2764-2768；Kim etc. (1994a) Proc.Natl.Acad.Sci.USA 91：883-887；Kim etc. (1994b) J.Biol.Chem.269：31,978-31,982.Thus, in one embodiment, fusion protein is included from least one (it can be changed for the cutting domain (or cutting half domain) of IIS type restriction enzymes and one or more Zinc finger binding domains It is making or do not transform).

It is Fok I that cutting domain and binding structural domain are the exemplary IIS types restriction enzymes that can divide.This specific enzyme is made It is activated for dimer.Bitinaite etc. (1998) Proc.Natl.Acad.Sci.USA95：10,570-10,575.Cause And, for purposes of this disclosure, it is believed that the Fok I enzymes part used in disclosed fusion protein is cutting half domain.Such as This, for the cutting of targeting double-strand and/or targeting that are carried out to cellular sequences using the ZFN-Fok I fusions comprising zinc finger are replaced Change, it is possible to use two fusion protein (each cuts half domain comprising Fok I) are rebuilding the knot of the cutting with catalysis activity Structure domain.Or, it is also possible to using the single polypeptide point comprising a Zinc finger binding domain and two Fok I cutting half domains Son.Carried out using zinc finger-Fok I fusions targeting cutting and targeting sequence change parameter be provided in the disclosure other places and For example U.S. Patent Application Publication text No.2005/0064474, disclosures of which is included by referring to.

In other embodiment, Fok I cutting half domain may include at one or many places it is any affect dimerization ammonia Mutation at base acid residue.Such mutation can be used for one of a pair of ZFP/Fok I fusions of prevention experience can be caused be not intended to The homodimerization cut at sequence.For example, the 446th of Fok I, the 447th, the 479th, the 483rd, the 484th, 486th, the 487th, the 490th, the 491st, the 496th, the 498th, the 499th, the 500th, the 531st, the 534th Position, the 537th and the 538th amino acids residue are close enough with dimerization interface affecting dimerization.Thus, can use The aminoacid sequence of the above-mentioned position at one or many places changes to change the dimeric nature of cutting half domain.Can be for example, by Build the variant of library and selection with desired characteristic for containing (or coding) different aminoacids residue in these positions, Huo Zhetong Cross the indivedual mutants of appropriate design to introduce such change.Outside homodimerization is prevented, it is also possible to, and using two open countries The cutting efficiency that raw type cutting half domain is obtained is compared, and some in these mutation can improve cutting efficiency.

Thus, for one or both of the targeting cutting carried out using a pair of ZFP/Fok I fusions, fusion protein can be wrapped Containing the following aminoacid change at one or many places, it suppresses self dimerization (self-dimerization) but allows two kinds The heterodimerization of fusion protein so that cut in the target site wanted.In certain embodiments, change and be present in two kinds In fusion protein, and change with synergistic effect；That is, make to cause arbitrary fusions homodimerization of abnormal cutting to minimize or Eliminate, and compared with the heterodimerization that half domain is obtained is cut using wild type, promote the different of described two fusion protein Dimerization.

In certain embodiments, cutting domain cuts half domains (both comprising integrated structure comprising two A part for the single polypeptide in domain), i.e., the first cutting half domain and second cuts half domain.Cutting half domain can have Identical aminoacid sequence or different aminoacid sequences, as long as they play the function of cutting DNA.

Cutting half domain can be with the offer in separate molecule.For example, two kinds of fused polypeptide can be expressed in cell, Wherein every kind of polypeptide includes binding structural domain and cutting half domain.Cutting half domain can have identical aminoacid sequence or Different aminoacid sequence, as long as they play the function of cutting DNA.Additionally, binding structural domain combines target sequence, it is typically It is arranged in such a way so that after fused polypeptide is combined, two cutting half domains (are for example led to allowing that cutting domain is rebuild Cross the dimerization of half domain) spatial orientation each other present, thus half domain is relative to each other placed to form function Property cutting domain, causes the cutting to cyto-chromatin in area-of-interest.In general, by the cutting domain institute for rebuilding The site that the cutting for carrying out is occurred between two target sequences.One or both of protein can be transformed into reference to its target Site.

The expression of two kinds of fusion protein in cell can be derived from and for described two protein be delivered to the cell；By one A kind of delivering nucleic acid of one of protein and code for said proteins is planted to the cell；By two kinds of each code for said proteins it One delivering nucleic acid is to the cell；Or by the way that the single nucleic acid for encoding two kinds of protein is delivered to into the cell.Other In embodiment, fusion protein includes wall scroll polypeptide chain, and it includes two cutting half domains and a Zinc finger binding domain. In this case, single fusion protein is expressed in cell, and (on the premise of being not intended to be bound by theory) thinks it Due to formed cutting half domain molecule interdimers and cutting DNA.

In certain embodiments, by the member array of ZFP into cause Zinc finger domain apart from fusion protein aminoterminal Recently, and cause cutting half domain it is nearest apart from c-terminuses.Which reflects naturally occurring dimerization cutting domain (such as Those are derived from Fok I enzymes) in cutting domain relative orientation, wherein DNA binding structural domains are nearest apart from amino terminal And it is nearest apart from carboxyl terminal to cut half domain.In these embodiments, cut half domain dimerization and form function Property nuclease be by fusion protein be bound to opposition DNA on site and cause, wherein binding site 5 ' end each other It is close to.

In this orientation, the close Fok I of zinc finger of most C-terminal cut half domain.Previously it has been determined that in CCHC type zinc In the presence of referring to so that most C-terminal refers to, non-standard zinc finger protein most efficiently combines its DNA target thing.Therefore, it is possible that, with Fok I The presence of the close previously described CCHC types zinc finger of cutting half domain suppresses its function.If situation thus, if the disclosure Jing The CCHC zinc fingers for crossing optimization obviously lie in less than the inhibitory activity of this hypothesis.

In other embodiment, by the member array of fusion protein (such as ZFP-Fok I fusions) into so that cutting Half domain is nearest apart from the amino terminal of fusion protein and Zinc finger domain is nearest apart from carboxyl terminal.In these embodiments In, it is the site being bound to by fusion protein on opposition DNA to cut half domain dimerization and form functional nucleic acid enzyme And cause, 3 ' ends of wherein binding site are closer to each other.

In other embodiment, the first fusion protein is included apart from the nearest cutting of the amino terminal of the fusion protein half Domain and apart from the nearest Zinc finger domain of carboxyl terminal, and the second fusion protein is arranged so that into Zinc finger domain distance The amino terminal of the fusion protein is recently and cutting half domain is nearest apart from carboxyl terminal.In these embodiments, two kinds Fusion protein combines identical DNA, wherein comprising the first fusion protein apart from the nearest Zinc finger domain of carboxyl terminal Binding site is located at 5 ' sides of the binding site comprising the second fusion protein apart from the nearest Zinc finger domain of amino terminal.Also WO 2005/084190 is can be found in, by referring to the disclosure of which is included.

Aminoacid sequence between Zinc finger domain and cutting domain (or cutting half domain) is referred to as " ZC joints ".ZC Joint is otherwise varied between joint and finger mentioned above.Details with regard to obtaining the ZC joints of optimizing incision see, for example, United States Patent (USP) Disclosure 20050064474A1 and 20030232410, and International Patent Publication text WO 2005/084190, by referring to And include disclosures of which.

Expression vector

The nucleic acid clone of one or more ZFP or ZFP fusion protein (such as ZFN) of coding can be entered in carrier, to It is transformed in prokaryotic cell or eukaryotic cell to replicate and/or express.Carrier can be prokaryotic vector or eukaryotic vector, including but It is not limited to plasmid, shuttle vector, insect vector, binary vector and (see, for example, United States Patent (USP) No.4,940,838；Horsch etc. (1984)Science 233：496-498, and (1983) Proc.Nat ' l.Acad.Sci.USA 80 such as Fraley：4803) etc.. The nucleic acid clone of coding ZFP can also be entered in expression vector, for applying to plant cell.

For expressed fusion protein, typically the sequence of coding ZFP or ZFP fusions is subcloned into containing guidance transcription Promoter expression vector in.Suitable antibacterial and eukaryotic promoter are well known in the art, and are recorded in for example Sambrook etc., Molecular Cloning, A Laboratory Manual (second edition, 1989；3rd edition, 2001)； Kriegler, Gene Transfer and Expression：A LaboratoryManual(1990)；And Current Protocols in Molecular Biology (Ausubel etc., see above).In such as escherichia coli (E.coli), spore The bacterial expression system for expressing ZFP can be obtained in bacillus (Bacillus sp.) and Salmonella (Salmonella) (Palva etc., Gene22：229-235(1983)).The test kit of these expression systems is commercialization.It is thin for mammal The eukaryotic expression system of born of the same parents, yeast and insect cell is well known to the skilled person, and is also commercialization.

Promoter for instructing the expression of nucleic acid of coding ZFP depends on specific application.For example, typically using suitable Express in the strong constitutive promoter of host cell and purification ZFP.

Comparatively, apply ZFP in vivo (see below " by delivering nucleic acid to plant cell " during plant gene with being adjusted Part), using composing type or inducible promoter, this depends on the special-purpose of ZFP.The non-limitative example of plant promoter Including from promoter sequence derived from the following group：Arabidopsiss (A.thaliana) ubiquitin -3 (ubi-3) (Callis etc., 1990, J.Biol.Chem.265：12486-12493)；Agrobacterium tumefaciens (A.tumifaciens) mannopine synthase (Δ mas) (Petolino etc., United States Patent (USP) No.6,730,824)；And/or cassava vein mosaic virus (CsVMV) (Verdaguer etc., 1996, Plant Molecular Biology 31：1129-1139).Referring also to embodiment.

Outside promoter, expression vector typically comprises transcriptional units or expression cassette, its be included in host cell (or It is protokaryon or eucaryon) in all other element needed for express nucleic acid.Thus, typical expression cassette is included and for example encoded The promoter that the nucleotide sequence of ZFP is operatively connected, and required signal, for example for effective polyadenylation of transcript, Tanscription termination, ribosome binding site or translation termination.Other element of the box can include such as enhancer, and heterologous cut Connect signal.

According to the intended purpose of ZFP, for example, express to select to use in plant, animal, antibacterial, funguses, protozoacide etc. In the particular expression carrier (see below the expression vector) for hereditary information being transported in cell.The antibacterial and animal of standard Expression vector is well known in the art, and is recorded in such as United States Patent (USP) disclosure 20050064474A1 and state in detail Border Patent Publication WO05/084190；WO05/014791；And WO03/080809, their disclosure is included by referring to Content.

Can be produced using Standard transfection methods express a large amount of protein antibacterial, mammal, yeast or insecticide it is thin Born of the same parents are then can to carry out protein described in purification using standard technique and (see, for example, Colley etc., J.Biol.Chem.264： 17619-17622(1989)；Guide to Protein Purification, in Methods in Enzymology, volume 182 (Deutscher is compiled, 1990)).The conversion of eukaryotic cell and prokaryotic cell is implemented (to see, for example, according to standard technique Morrison, J.Bact.132：349-351(1977)；Clark-Curtiss and Curtiss, Methods in Enzymology 101：347-362 (Wu etc. is compiled, 1983)).

Can be using any known code for importing foreign nucleotide sequences in such host cell.These include Using calcium phosphate transfection, Polybrene, protoplast fusion, electroporation, ultrasonic method (such as sound perforation (sonoporation)), liposome, microinjection, naked DNA, plasmid vector, viral vector are (additional build and integrated Two kinds) and it is any thin for the genomic DNA cloned, cDNA, synthetic DNA or other foreign heredity substances to be imported into host Other well known method (see, for example, Sambrook etc., see above) in born of the same parents.Uniquely it is essential that the specific hereditary work for being used Journey code successfully can express at least one channel genes in the host cell of selected protein.

By delivering nucleic acid to plant cell

As described above, DNA construct can be imported by various routine techniquess and (for example led in desired plant host In entering its genome).For the summary of such technology, Weissbach and Weissbach, Methods for is see, for example, Plant Molecular Biology (1988, Academic Press, N.Y.) save VIII, page 421-463；And Grierson And Corey, Plant Molecular Biology (1988, second edition), Blackie, London, chapter 7-9.

For example, DNA construct can be led using the technology such as electroporation and microinjection of plant cell protoplast In entering plant cell, or DNA construct can be introduced directly into plant group using biolistic methods (biolisticmethod) In knitting, DNA particle bombardment (see, for example, Klein etc. (1987) Nature 327：70 73).Or, DNA construct can To combine with suitable T-DNA flanking regions, and import in conventional Agrobacterium tumefaciens host cell.Agrobacterium tumefaciens mediate Transformation technology (using and eliminating (disarming) including binary vector) be recorded in completely in scientific literature.See, for example, Horsch etc. (1984) Science 233：496498, and (1983) Proc.Nat ' l.Acad.Sci.USA80 such as Fraley： 4803。

Additionally, gene transfer can use the antibacterial of non-agrobacterium category or virus (such as Rhizobium sp NGR234 Take root in (Rhizobium sp.NGR234), Sinorhizobium meliloti (Sinorhizoboium meliloti), Radix Loti Corniculati tumor Bacterium (Mesorhizobium loti), potato virus X, cauliflower mosaic viruses and cassava vein mosaic virus and/or Nicotiana tabacum L. Mosaic viruss) realizing.See, for example, Chung etc. (2006) Trends Plant Sci.11 (1)：1-4.

The virulence function of Agrobacterium tumefaciens host can be using binary T DNA vector (Bevan (1984) Nuc.Acid Res.12：8711-8721) or co-culture code (Horsch etc. (1985) Science227：1229-1231) by the antibacterial sense Contaminate and instruct after the cell in construct and proximity marker insertion plant cell dna.In general, being turned using Agrobacterium Change system is transforming dicotyledon (Bevan etc. (1982) Ann.Rev.Genet 16：357-384；Rogers etc. (1986) MethodsEnzymol.118：627-641).Agrobacterium conversion system can be also used for conversion and transfer DNA to list Leaf plant and plant cell.Referring to United States Patent (USP) No.5,591,616；Hernalsteen etc. (1984) EMBO J 3：3039- 3041；Hooykass Van Slogteren etc. (1984) Nature 311：763-764；Grimsley etc. (1987) Nature 325：1677-179；Boulton etc. (1989) Plant Mol.Biol.12：31-40；And (1991) Plant such as Gould Physiol.95：426-434.

Alternative gene transfer and method for transformation is including but not limited to mediated by calcium, Polyethylene Glycol (PEG) or electroporation The naked DNA protoplast transformation that carries out of intake (referring to Paszkowski etc. (1984) EMBO J 3：2717-2722； Potrykus etc. (1985) Molec.Gen.Genet.199：169-177；Fromm etc. (1985) Proc.Nat.Acad.Sci.USA 82：5824-5828；And Shimamoto (1989) Nature 338：274-276) and plant Electroporation (D ' Halluin etc. (1992) the Plant Cell4 of tissue：1495-1505).For other side of transforming plant cells Method includes DNA intake (Kaeppler etc. (1990) the Plant Cell Reporter 9 that microinjection, carborundum are mediated：415- And microparticle bombardment is (referring to Klein etc. (1988) Proc.Nat.Acad.Sci.USA 85 418)：4305-4309；And Gordon- Kamm etc. (1990) Plant Cell 2：603-618).

Disclosed method and composition can be used for for exogenous array inserting position predetermined in plant cell gene group. This is useful, because the expression for importing the transgenic in Plant Genome is critically depend on its integration site.Thus, encode example Gene such as nutrient, antibiotic or therapeutic molecules can be recombinated by targeting and insert in Plant Genome and be conducive to it to express Region.

The conversion plant cell produced by any of above transformation technology can be cultivated and possess converted gene to regenerate Type and it is therefore desirable for phenotype whole plant.Such regeneration techniques depends on certain plants in tissue culture growth media to swash The operation of element, typically depends on the biocide and/or herbicide mark for importing together with desired nucleotide sequence Will thing.Evans etc. is recorded in from the protoplast regeneration plant of culture, " Protoplasts Isolation and Culture " is in Handbook of Plant Cell Culture, pp.124-176, Macmillian Publishing Company, New York, 1983；And Binding, Regeneration of Plants, Plant Protoplasts, Pp.21-73, CRC Press, Boca Raton, 1985.Can be with from plant corpus callosum (callus), explant, organ, flower Powder, embryo or part thereof are regenerated.Such regeneration techniques is recorded in general manner Klee etc. (1987) Ann.Rev.of Plant Phys.38：467-486.

The nucleic acid imported in plant cell can be used to give substantially any plant with anticipant character.The disclosure can be used Nucleic acid construct and above-mentioned various method for transformation to transform expectation described herein to extremely various plants and plant cell system Physiology and agronomic characteristics.In certain embodiments, the target plant and plant cell for transformation includes being not limited to that A little unifacial leaves and dicotyledon, such as crops, it includes cereal crops (such as Semen Tritici aestivi, Semen Maydiss, rice, broomcorn millet (millet), big Wheat), fruit crop (such as Fructus Lycopersici esculenti, Fructus Mali pumilae, pears, Fructus Fragariae Ananssae, Citrus chachiensis Hort./Fructus Citri tangerinae/orange), forage crop (such as Herba Medicaginiss), root vegetables crop (rootvegetable crop) (such as Radix Dauci Sativae, Rhizoma Solani tuber osi, Radix Betae, Rhizoma Dioscoreae), leaf vegetables crop (leafy Vegetablecrop) (such as Caulis et Folium Lactucae sativae, Herba Spinaciae)；Flowering plant (such as petunia, Flos Rosae Multiflorae/Flos Rosae Rugosae, chrysanthemum), conifer and pine Tree (such as fir (pine fir), PiceameyeriRehd. Et Wils.)；For plant (such as heavy metal product of phytoremediation (phytoremediation) Tired plant (heavy metal accumulating plant))；Oil crop (such as Helianthi, Semen Brassicae campestriss) and for testing The plant (such as Arabidopsises) of purpose.Thus, disclosed method and composition has purposes on extensive plant, it includes But it is not limited to the species from such as subordinate：Asparaguses (Asparagus), Avena (Avena), Btassica (Brassica), Citrus (Citrus), Citrullus (Citrullus), Capsicum (Capsicum), Cucurbita (Cucurbita), Daucuss (Daucus), Glycine (Glycine), Gossypium (Gossypium), Hordeum (Hordeum), Lactuca (Lactuca), Fructus Lycopersici esculenti Category (Lycopersicon), Maluses (Malus), cassava (Manihot), Nicotiana (Nicotiana), Oryza (Oryza), Persea (Persea), Pisum (Pisum), pear (Pyrus), Prunus (Prunus), Rhaphanuss (Raphanus), Secale (Secale), Solanum (Solanum), sorghum (Sorghum), Triticum (Triticum), Vitiss (Vitis), Vigna And Zea (Zea) (Vigna).

Those skilled in the art can approve, stably be mixed in transgenic plant in expression cassette and confirm it is operable After, it can be imported in other plants by sexual hybridization.Can be using any one of many standard breeding techniques, this takes Certainly in species to be hybridized.

Inverted plant cell, corpus callosum, tissue or plant by selecting improved vegetable material or can sieve The character by present on converted DNA coded by marker gene is selected to be identified and isolated from.For example, selection can be implemented as follows, Will improved vegetable material cultivate in the culture medium containing amount of suppression antibiotic or herbicide, institute's transformed gene construct Give to the antibiotic or the resistance of herbicide.Additionally, inverted plant and plant cell can also can be with by screening Any visible mark gene (such as β-glucuronidase, luciferase, B or the C1 being present in recombinant nucleic acid construct Gene) activity identifying.Such selection and screening technique are well known to the skilled person.

The plant or plant of the gene construct containing having inserted can also be identified using physics and biochemical method Cell transformation.These methods are included but is not limited to：1) Sourthern analyses or PCR amplifications, for detecting and determining restructuring The structure of DNA inserts；2) Northern traces, S 1RNA enzyme protections, primer extension or reverse transcription-PCR amplification, for examining Survey and check the RNA transcript of gene construct；3) enzyme assay, for detecting enzyme or ribozyme activity, wherein this genoid is produced Thing is encoded by gene construct；4) gel electrophoresis of protein, western blot technology, immunoprecipitation or enzyme-linked immunoassay Method, wherein gene construct product are protein.Can also be using other technology (such as in situ hybridization, enzyme dyeing and immunity dye Color) come detect recombinant precursor specified plant organ and tissue in presence or expression.For carrying out all these algoscopys Method be well known to the skilled person.

The effect of the genetic manipulation carried out using presently disclosed method can be for example, by self-interested separate tissue The Northern traces of RNA (such as mRNA) are observing.Typically, if the amount of mRNA increased, then it is considered that corresponding Endogenous gene just reached with the more faster Speedometer Drive than before.Can use measurement base because and/or active other methods of CYP74B. Different types of enzyme assay can be used, is increased or decreased depending on the substrate and detection product or by-product for using Method.In addition, the level of expressed protein and/or CYP74B albumen can be measured by immuno-chemical method, i.e., ELISA, RIA, EIA and for well known to a person skilled in the art other based on antibody algoscopys, such as by electrophoresis detection Algoscopy (or using dye or use western blot).Transgenic can be in some tissues of plant or at some Stage of development selective expression, or transgenic can be in essentially all of plant tissue, substantially in its whole life Express in cycle.However, any combinations expression pattern is also applicable.

The disclosure is also contemplated by the seed of transgenic plant mentioned above, wherein the seed has transgenic or gene constructed Body.The disclosure is also contemplated by offspring, clone, cell line or the cell of above-mentioned transgenic plant, wherein the offspring, clone, cell System or cell have the transgenic or gene construct.

The expression vector of ZFP and coding ZFP can be applied directly to plant, cut for targeting and/or recombinated.

The administration of effective dose is generally used for any path for importing ZFP and finally contacting with pending plant cell Carry out.ZFP is applied in any suitable manner.The appropriate method for applying such composition is obtainable, and for ability Field technique personnel are known, although and a kind of path can be used more than applying specific compositionss, it is specific Path generally can provide than another kind of path more in time (immediate) and more effectively reaction.

Carrier can also be used, and its part is according to the particular composition applied, and according to being used to apply this The ad hoc approach of compositionss is determining.Thus, there are the extremely various suitable formulation in pharmaceutical compositions that can be used (referring to example Such as Remington ' s Pharmaceutical Sciences, the 17th edition, 1985)).

Using

Zinc finger protein comprising one or more non-standard zinc fingers described herein can be used for current operating specification C2H2ZFP and enter The capable regulation and control of all genomes and editor are applied, including but not limited to：Gene activation；Gene is prevented；Genome editor (cutting, Targeting insertion, replacement are deleted)；With exopathogenic factor genome editor (via histone or the targeting of the covalent modification of DNA).

ZFN comprising non-standard zinc finger disclosed herein can be used for the DNA in incising cell chromatin at area-of-interest (such as site want in genome or predetermined, such as in the gene of saltant type or wild type).For such target To DNA cuttings, Zinc finger binding domain is transformed into reference to predetermined cuts site or neighbouring target site, and is expressed in cell Fusion protein comprising through engineering approaches Zinc finger binding domain and cutting domain.Target position is bound in the zinc finger part of fusion protein Point after, by cutting domain near target site cutting DNA.The exact site of cutting may depend on the length of ZC joints.

Or, two kinds of respectively ZFN comprising Zinc finger binding domain and cutting half domain are expressed in cell, and be bound to Target site, it is so that the mode that feature cutting domain is able to rebuild and DNA is able to cut near target site is arranged side by side. In one embodiment, cutting occurs between the target site in two Zinc finger binding domains.One of Zinc finger binding domain or The two can be transformed.

For the targeting cutting carried out using Zinc finger binding domain-cutting domain fused polypeptide, binding site can be with Cover cleavage site, or the proximal edge of binding site can apart from cleavage site 1,2,3,4,5,6,10 Individual, 25,50 or more polynucleotide (or any integer value between 1 and 50 nucleotide).Binding site is relative The length of specific cutting domain and ZC joints can be depended in the accurate location of cleavage site.It is each for wherein using two kinds Method comprising Zinc finger binding domain and the fusion protein of cutting half domain, binding site typically crosses over cleavage site.Such as This, the proximal edge of the first binding site can be 1,2,3,4,5,6,10,25 of cleavage site side Or more polynucleotide (or any integer value between 1 and 50 nucleotide), and the proximal edge of the second binding site can Be 1,2,3,4,5,6,10,25 of cleavage site opposite side or more polynucleotide (or between 1 and Any integer value between 50 nucleotide).For in vitro and in vivo to the method for cleavage site mapping to this area skill Art personnel are known.

Once importing in target cell or expressing in target cell, fusion protein is bound to target sequence, and at target sequence or Nearby cut.The exact site of cutting depends on the property of cutting domain and/or combines and cutting domain between The presence of joint sequence and/or property.In the case of respectively including the ZFN of cutting half domain using two kinds wherein, bound site The distance between proximal edge of point can be 1,2,3,4,5,6,7,8,9,10,25 or more Nucleotide (or any integer value between 1 and 50 nucleotide).The optimum level of dissection might also depend on The distance between binding site of two ZFN (see, for example, Smith etc. (2000) Nucleic Acids Res.28：3361- 3369；Bibikova etc. (2001) Mol.Cell.Biol.21：Both length of ZC joints 289-297) and in each ZFN.Also Can be found in United States Patent (USP) disclosure 20050064474A1 and International Patent Publication text WO 05/084190；WO 05/ 014791；With WO 03/080809, disclosures of which is included by referring to.

Two ZFN respectively comprising cutting half domain can be tied in the region of interest with the polarity of identical or opposition Close, and their binding site (i.e. target site) can by any number of nucleotide (such as from 0 to 50 nucleotide pair or Any integer value therebetween) separate.In certain embodiments, two respectively include Zinc finger binding domain and cutting half domain Fusion protein binding site can at a distance of between 5 and 18 nucleotide pairs, such as at a distance of 5-8 nucleotide pair, Or at a distance of 15-18 nucleotide pair, or at a distance of 6 nucleotide pairs, or 16 nucleotide pairs apart, or 10 nucleoside apart Acid within, as measured by according to each binding site with the immediate edge of another binding site, and cut generation exist Between binding site.

The site that DNA carries out cutting place is normally between the binding site of two fusion protein.The double-strand of DNA is broken Split the single-strand break for generally originating from two skews (offset) 1,2,3,4,5,6 or more polynucleotide (break) or " otch " (nick) (such as cuttings of the natural Fok I to double-stranded DNA is derived from the single-stranded disconnected of 4 nucleotide of skew Split).Thus, the site that cutting need not just oppose on every DNA occurs.In addition, the structure and target site of fusion protein Between distance can affect cutting whether adjacent to single nucleotide acid to occurring, or whether cutting occurs in several sites.However, for Many applications (including targeting restructuring and targeted mutagenesis), the cutting in certain limit nucleotide is usually enough, and is not required to Want the cutting between particular bases pair.

As described above, can express in cell one or more after polypeptide and/or polynucleotide being imported in cell Fusion protein.For example, the polynucleotide of two kinds of sequences respectively comprising one of encoding such polypeptides can be imported in cell, and Expression of polypeptides and each polypeptide is bound to after its target sequence, at target sequence or near cut.Or, will comprising coding two kinds The single polynucleotide of the sequence of fused polypeptide are imported in cell.Polynucleotide can be appointing for DNA, RNA or DNA and/or RNA What modified forms or the like.

In certain embodiments, the targeting cutting for being carried out by ZFN in genome area causes by nonhomologous end The change of the Region Nucleotide sequence after the cutting event is repaired in connection (NHEJ).

In other embodiments, the targeting cutting for being carried out by ZFN in genome area can also be the one of following code Part, in the code via homology dependent mechanism with sequence replacement gene group sequence that is homologous but differing (for example Area-of-interest in cyto-chromatin) (i.e. by targeting recombinate) (for example comprising exogenous array and one or more snippets with it is pre- Determine the insertion of the donor sequences of sequence that genome sequence (i.e. target site) is identical or homologous but differs).Because in cell DNA Double-strand break stimulate cellular repair mechanisms near cleavage site up to thousands of times, the targeting cutting carried out with ZFN described herein The sequence for allowing actually any site in genome changes or replaces (via the reparation that homology is instructed).

Outside ZFN described herein, the targeting of selected genes group sequence is replaced and also needs to import external source (donor) polynucleotide. Can be by donor polynucleotide before expression ZFN, while or importing afterwards in cell.Donor polynucleotide and genome sequence Row are containing enough homologys supporting it and have homologous recombination between the genome sequence of homology (or homology with it The reparation of guidance).About 25,50,100,200,500,750,1,000,1,500,2,000 nucleoside Acid or more sequence homology (or between 10 and 2, any integer value between 000 nucleotide, or more) can support together Recombinate in source.The length range of donor polynucleotide can be from 10 to 5,000 nucleotide (or any integer of nucleotide therebetween Value) or it is longer.

It is readily apparent that typically, the genome sequence that the nucleotide sequence of donor polynucleotide is replaced with it not phase Together.For example, the sequence of donor polynucleotide can containing one or more relative to the replacement of genome sequence, insertion, deletion, Inversion or rearrangement, as long as there is enough homologys with chromosome sequence.Such sequence variation can be any size, Er Qieke With little to single nucleotide acid pair.Or, donor polynucleotide can be comprising the nonhomologous sequence that flank is two homology regions (i.e. exogenous array, it is distinguished with exogenous polynucleotide).In addition, donor polynucleotide may make up carrier molecule, its contain with Area-of-interest does not have the sequence of homology in cyto-chromatin.In general, the homology region of donor polynucleotide and expectation weight The genome sequence of group can have at least 50% sequence iden.In certain embodiments, have 60%, 70%, 80%, 90%th, 95%, 98%, 99% or 99.9% sequence iden.There may be between 1% and 100% sequence iden Any numerical value, this depend on donor polynucleotide length.

Donor molecule can include the discontinuity zone that several and cyto-chromatin has homology.For example, for targeting Insertion is not present under normal circumstances the sequence in area-of-interest, and the sequence may reside in donor nucleic acid molecules, and And its flank is the region for having homology with the sequence in area-of-interest.

In order to simplify for determine from donor polynucleotide sequence succeed insertion algoscopy (for example hybridization, PCR, restriction enzymic digestion), there may be some sequence differences compared with genome sequence in donor sequences.Preferably, if In coding region, then such nucleotide sequence difference will not change aminoacid sequence, or the aminoacid that can produce silence Change (not affecting the change of protein structure or function).Donor polynucleotide can optionally comprising in area-of-interest with Change in the corresponding sequence of Zinc finger domain binding site, to prevent to importing by homologous recombination cyto-chromatin In donor sequences cutting.

Can import polynucleotide as a part for carrier molecule in cell, the carrier molecule has other sequence The gene of row, such as replication orgin, promoter and coding antibiotic resistance.Furthermore, it is possible to naked nucleic acid form, with reagent (such as liposome or Poloxamer) compound nucleic acid, or can be by antibacterial or disease importing donor polynucleotide Poison (such as raw root nodule bacteria in Agrobacterium, Rhizobium sp NGR234, Sinorhizobium meliloti, Radix Loti Corniculati, tobacco mosaic virus (TMV), Potato virus X, cauliflower mosaic viruses and cassava vein mosaic virus) delivering donor polynucleotide.See, for example, Chung Deng (2006) Trends Plant Sci.11 (1)：1-4.

For the change of chromosome sequence, it is not necessary to which the full sequence of donor is copied in chromosome, as long as the confession of copy Body sequence be enough to realize that desired sequence changes.

The efficiency of the donor sequences insertion carried out by homologous recombination ruptures with cell DNA double center chain and expectation is recombinated The distance between site be inversely proportional to.In other words, observe when double-strand break is nearer with the site for expecting to be recombinated higher Homologous recombination efficiency.(such as desired recombination event can be in one section of base in the case of accurate recombination site is not made a reservation for Because occurring in group sequence), the length and sequence to donor nucleic acid, and cleavage site selected to obtain desired restructuring thing Part.It is designed to change in genome sequence in the case of the sequence of single nucleotide acid pair, in that nucleotide in desired event To 10,000 nucleotide internal cutting cyto-chromatins of either side.In certain embodiments, in the core that sequence is to be altered The either side 1,000 of thuja acid pair, 500,200,100,90,80,70,60,50,40,30,20 Individual, 10,5 or 2 nucleotide, or between 2 and 1, cut in any integer value between 000 nucleotide.

Cut by using the targeting in the genome area that ZFN is carried out, while provide the external source comprising exogenous array (supplying Body) polynucleotide, realize exogenous array targeting being inserted in genome area.Typically, donor polynucleotide also includes external source The sequence of sequence flank, it be enough to support the homology that genome sequence double center chain ruptures with the homology that genome area contains The reparation of guidance, thus inserts exogenous array in genome area.Therefore, donor nucleic acid can be enough to support by homologous Property dependency repair mechanism (such as homologous recombination) and integrate exogenous array any size.It is not intended to be limited by any particular theory System, it is believed that the homology region of exogenous array flank provides to the end of chromosome of fracture is used for resynthesis double-strand break site The template of place's hereditary information.

The targeted integration of exogenous array mentioned above can be used to insert marker gene in selected chromosome position.Marker gene (for example amicillin resistance, neomycin resistance, G418 resistances, purine are mould including but not limited to encode mediated antibiotic resistance Plain resistance) protein sequence, encoding coloured or fluorescence or luminous protein (such as green fluorescent protein, strengthens Type green fluorescent protein, red fluorescent protein, luciferase), and the albumen for mediating enhanced cell growth and/or gene amplification The sequence of matter (such as dihydrofolate reductase).Thus, exemplary marker gene includes but is not limited to β-glucuronidase (GUS), the double oxygenations of phosphinothricin N acetyl transferring enzyme (PAT, BAR), neomycin phosphotransferase, beta-lactamase, catechol Enzyme, α-amylase, tryrosinase, beta galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, second Acyl lactic acid synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase.At some In embodiment, targeted integration can be used to insert rna expression construct, for example, be responsible for the modulated expression of Microrna or siRNA Sequence.Promoter, enhancer or other transcriptional regulatory sequences can also be mixed in rna expression construct.

The further increase of targeting recombination efficiency in cell comprising zinc finger/histone-nuclease fusion molecule and donor DNA molecule Be achieved by, will cells blocks cell cycle G₂Phase, the repair process that now homology drives has most Radix Angelicae Dahuricae (Radix Heraclei Scabridi) Property.This retardance can be achieved in many ways.For example, can be with medicine, the compound for for example affecting cell cycle progression And/or small molecule is processing cell so that by cell block in G₂Phase.Such exemplary molecule includes but is not limited to shadow The compound (such as vinblastine (vinblastine), nocodazole (nocodazole), Taxol (Taxol)) of sound microtubule polymerization, Compound (such as cis-platinum (II) diammine dichloro compound (cis-platinum (II) interacted with DNA Diaminedichloride), cisplatin (Cisplatin), doxorubicin (doxorubicin)) and/or affect DNA synthesis change (for example thymidine, hydroxyurea, L- .beta.-[N-(3-hydroxy-4-pyridone)s (L-mimosine), etoposide (etoposide), 5- fluorine urine are phonetic for compound Pyridine).Being further added by of recombination efficiency is achieved by, i.e., using the histone deacetylase (HDAC) for changing chromatin Structure Inhibitor (such as sodium butyrate, Atrichostatin A (trichostatin A)) is more easy to be recombinated machine by cell making genomic DNA (machinery) it is close to.

Method for distinguishing for cell cycle arrest includes that overexpression suppresses the protein of CDK cell-cycle kinases activity, For example by the way that the cDNA for encoding the protein is imported in cell or by by the gene expression of protein described in activated code Through engineering approaches ZFP import in cell to realize.Cell cycle arrest is also accomplished as, i.e., using such as RNAi methods (for example United States Patent (USP) No.6,506,559) suppressing the activity of cyclin and CDK, or by the way that following through engineering approaches ZFP are imported In cell, one or more gene being involved in the through engineering approaches ZFP inhibitory cell cycle progression (such as cell cycle Albumen and/or CDK genes) expression.The method of the through engineering approaches zinc finger protein expressed for regulator gene with regard to synthesis is referring to example Such as total United States Patent (USP) No.6,534,261.

As described above, the disclosed method and composition for targeting cutting can be used in induced gene group sequence Mutation.Targeting cutting can also be used to produce gene knockout (such as functional genomicses or target validation (target Validation)) and for promoting sequence targeting to insert in genome (i.e. gene knock-in).Insertion can be implemented as described below, that is, be led to Cross homologous recombination or chromosome sequence is replaced by targeted integration, wherein being homologous with area-of-interest in chromosome by flank The new sequence (i.e. non-existent sequence in area-of-interest) of sequence be inserted in predetermined target site.Identical method can also be used In wild-type sequence is replaced with mutant sequences, or for a kind of allele to be converted into into different allele.

Targeting cutting to phytopathogen that is being infected or being integrated can be used for treating the cause of disease in plant host Body-sensing contaminates, for example, carry out cutting by the genome to pathogen so that its pathogenicity reduces or eliminates to realize.In addition, to compiling The targeting cutting of the gene of code plant viruses receptor can be used to block the expression of this receptoroid, thus prevent the virus sense in plant Dye and/or virus disseminating.

Exemplary phytopathogen includes but is not limited to plant viruses, such as Alfamovirus (Alfamovirus), Alphacryptovirus (Alphacryptovirus), Badnavirus (Badnavirus), β hide disease Poison category (Betacryptovirus), Bigeminivirus, bromovirus (Bromovirus), Fructus Hordei Vulgaris yellow mosaic disease Poison category (Bymovirus), capillovirus category (Capillovirus), Dianthovirus (Carlavirus), xiangshizhubanbo Tobamovirus (Carmovirus), cauliflower mosaic viruses group (Caulimovirus), long closterovirus group (Closterovirus), cowpea mosaic virus group (Comovirus), cucumber mosaic virus group (Cucumovirus), matter type bullet Shape Tobamovirus (Cytorhabdovirus), dianthovirus category (Dianthovirus), Bowl bean lug mosaic viruss group (Enamovirus), broad bean wilt virus group (Fabavirus), Fijivirus category (Fijivirus), furovirus group (Furovirus), Hordeivirus (Hordeivirus), Hybrigeminivirus, Fructus Rubi corchorifolii Immaturuss Tobamovirus (Idaeovirus), annulus orae group (Ilarvirus), sweet potato mild mottle virus category (Ipomovirus), Fructus Hordei Vulgaris are yellow Dwarf virus group (Luteovirus), Machomovirus (Machlomovirus), Macluravirus (Macluravirus), Marafivirus (Marafivirus), Monogeminivirus, dwarf virus category (Nanavirus), Necrovirus (Necrovirus), nematicide pass polyhedrosis viruses group (Nepovirus), caryogram rhabdoviruses Category (Nucleorhabdovirus), Oryzavirus (Oryzavirus), Ourmiavirus, Phytoreovirus (Phytoreovirus), potato virus X group (Potexvirus), Potyvirus group (Poteyvirus), rye (Secale cereale L.) showy flowers of herbaceous plants Mosaic virus category (Rymovirus), satellite RNA, associated viruss (satellivirus), associated virus category (Sequivirus), south Square adzuki bean mosaic virus group (Sobemovirus), very thin Tobamovirus (Tenuivirus), tobacco mosaic virus (TMV) group (Tobamovirus), Tobacco rattle virus group (Tobravirus), tomato bushy stunt virus group (Tombusvirus), Fructus Lycopersici esculenti speckle wither Tobamovirus (Tospovirus), Trichovirus (Trichovirus), turnip yellow mosaic virus group (Tymovirus), umbrella shape are planted Thing Tobamovirus (Umbravirus), Varicosavirus (Varicosavirus) and stunt virus category (Waikavirus)；Mycosises Substance such as smut (such as Ustilaginales (Ustilaginales)), rest fungus (Uredinales (Uredinales)), Clavicipitaceae It is (Clavicepts pupurea) and mould；Mycete (Oomycete (Oomycetes)), such as phytophthora infestans (Phytophthora infestans) (potato blight)；Bacterial pathogens, such as Erwinia (Erwinia) are (for example The raw Erwinia (E.herbicola) of grass), Rhodopseudomonass (Pseudomonas) (such as Pseudomonas aeruginosa (P.aeruginosa), pseudomonas syringae (P.syringae), pseudomonas fluorescens (P.fluorescense) and stench are false Zymomonas mobiliss (P.putida)), Ralstonia (such as R.solanacearum), Agrobacterium (Agrobacterium) and Huang Zygosaccharomycess (Xanthomonas)；Nematicide (Nematoda (Nematoda))；And Phytomyxea (Polymyxa and root kind Pseudomonas (Plasmodiophora))。

The disclosed method for targeting restructuring can be used to replace any genome sequence with sequence that is homologous but differing Row.For example, mutated genes group sequence can be replaced with its wild type counterparts (counterpart), thus be provided for treating The method of plant disease；Resistance to phytopathogen is provided；Improve crop yield etc..Similarly, a kind of equipotential of gene Gene can be replaced with different allele, be carried out using targeting recombination method disclosed herein.

In many these situations, area-of-interest includes mutation, and donor polynucleotide includes corresponding wild type sequence Row.Similarly, wild-type genomic sequence can be replaced with mutant sequences, if it is desired to like this.Really, with any side Formula depends on any pathology of specific gene group sequence to be corrected or be relaxed using method disclosed herein and compositionss.

Targeting cuts and targeting restructuring can be also used for changing non-coding sequence (such as regulatory sequence, such as promoter, increasing Hadron, initial son, terminator, splice site) changing the expression of gene outcome.Such method can be used for for example treating Purpose, cytophysiology and biochemical change, functional genomicses and/or target validation research.

Methods described herein and compositionss can be additionally used in activation and suppressor gene expression, and it uses non-standard zinc finger to combine knot Fusions between structure domain and functional domain are realizing.Such method is disclosed in for example total United States Patent (USP) 6,534,261；6,824, 978；With 6,933,113, disclosures of which is included by referring to.It is other to prevent method including targeting gene to be prevented The use of the antisense oligonucleotide and/or siRNA (siRNA or RNAi) of sequence.

In other embodiment, outside zinc finger disclosed herein-cutting domain fusions or replace this paper institutes One kind between disclosed zinc finger-cutting domain fusions, Zinc finger binding domain and recombinase (or its functional fragment) Or various fusions can be used to promote targeting restructuring.See, for example, total United States Patent (USP) No.6,534,261 and Akopian etc. (2003)Proc.Natl.Acad.Sci.USA100：8688-8691.

In other embodiment, disclosed method and composition can be used to provide ZFP binding structural domains and transcription swashs The activity of the fusions of living or repression domain, the transcriptional activation or repression domain needs dimerization (or homodimerization Or heterodimerization).In such cases, fused polypeptide includes Zinc finger binding domain and functional domain monomer (such as from two The monomer of aggressiveness transcriptional activation or repression domain).Two such fused polypeptide combined with the target site of appropriate positioning allow into Row dimerization so that Reconstruction of The Function transcriptional activation or repression domain.

Embodiment

The present invention is explained further in the following embodiments, wherein unless otherwise mentioned, all fractions and percentage ratio are pressed Weight meter, and temperature presses celsius temperature scale meter.Although it should be appreciated that these embodiments specify some embodiment party of the present invention Case, but it is only illustratively given.From discussion above and these embodiments, those skilled in the art can determine that the present invention's Substitutive characteristics, and on the premise of without departing from the spirit and scope, various changes and modification can be carried out to the present invention so that its It is suitable to various uses and condition.

Embodiment 1：ZFN expression vectors

Following modification United States Patent (USP) disclosure 2005/0064474 (disclosure of which is included by referring to) (referring to The embodiment 2 of this application) embodiment 2 and 14 described in comprising coding 4 finger ZFN (referred to as " 5-8 " and " 5-9 ") sequence Expression vector.In short, by 5-8 and 5-9ZFN, (it is included via the ZC joints and IIS type restriction enzyme Fok I of 4 aminoacid Nuclease domain (Wah etc. (1998) Proc.Natl.Acad.Sci.USA 95：The 384- of the sequence of 10564-10569 579 amino acids) fusion 4 Zinc finger domains) be modified to CCHC structures.Also between C-terminal His and Cys zinc coordination structures Residue and/or refer to 2 and/or refer to that the 4 C-terminal residue of C-terminal Cys carries out other modification (substitute and insert).

Embodiment 2：The intergenic suppression of eGFP in reporting cell line

In Urnov (2005) Nature 435 (7042)：646-51 and United States Patent (USP) disclosure No.20050064474 ZFN of the test bag containing CCHC zinc fingers described herein promotes the ability of homologous recombination in (such as embodiment 6-11) described GFP systems. In short, according to the schemes of Invitrogen Lipofectamine 2000, each sample uses 2 μ L Lipofectamine The promoterless GFP donors of 50ng every kind of ZFN and 500ng (Urnov (2005) Nature) are transfected into 500,000 reports by 2000 In road cell.

The vinblastine of the 0.2 μM of final concentration of addition in 24 hours after transfection, and remove within 72 hours after transfection.

40,000 cells were measured come to cell by each transfection on the desk-top facs analysis instrument of Guava in 5 days after transfection Determine GFP expression.

As shown in figure 1, the most of ZFN comprising CCHC zinc fingers altered shown in table 1 above and 2 promote report (GFP) Homologous recombination at locus, result in higher than unmodified CCHC zinc fingers level GFP expression, and the performance of several ZFN with ZFN comprising CCHH zinc fingers is suitable.The optimal variant of performance is when being placed in finger 4 (F4) comprising following sequence (in His zinc The C-terminal of coordination residue is simultaneously coordinated residue including His zinc)：HAQRCGLRGSQLV(SEQ ID NO：53) (in table 2 be referred to as #21 and With the zinc finger shown in " 2-21 " in Fig. 1).The optimal variant of performance when being placed in finger 2 (F2) comprising following sequence ( The C-terminal of His zinc coordination residue is simultaneously coordinated residue including His zinc)：HIRTCTGSQKP(SEQ IDNO：75) (it is referred to as #43 in table 2 And with the zinc finger shown in " 2-43 " in Fig. 1).

Embodiment 3：By targeting restructuring editor's chromosome IL2R γ genes

Also in Urnov (2005) Nature 435 (7042)：646-51 and United States Patent (USP) disclosure ZFN described herein is determined in endogenous IL2R γ algoscopys described in the embodiment 2 of No.20050064474.In short, using Nucleofector (Amaxa) is transfected into the every kind of ZFN expression construct of 2.5 μ g in 500,000 K562 cells.Harvest gene Group DNA, and gene disruption is determined at endogenous IL2R γ locus using Surveyor endonuclease reagents box.

The upper left quarter of Fig. 2 shows ZFN.Specifically, altered zinc finger 20 refers to comprising sequence HTRRCGLRGSQLV CCHC zinc fingers；Zinc finger 21 includes sequence HAQRCGLRGSQLV (SEQ ID NO：53)；Zinc finger 43 includes sequence HIRTCTGSQKP (SEQ ID NO：75)；Zinc finger 45 includes sequence HIRTGCTGSQKP；Zinc finger 47 includes sequence HIRRCTGSQKP；And zinc finger 48 Comprising sequence HIRRGCTGSQKP.The zinc finger 20 and 21 used in the finger 4 of the ZFN of 4 fingers, and in the finger 2 of the ZFN of 4 fingers Using zinc finger 43,45,47 and 48.

In figure 2 above and in the right side of the figure and displayed in Table 5 ZFN pair for being tested：

Table 5

In order to determine whether the induced mutation at cleavage site, using Cel-1 algoscopys analysing amplified product is carried out, By amplified production degeneration in the Cel-1 algoscopys, and renaturation, then with mispairing specific C el-1 nucleic acid ferment treatments.Referring to example Such as Oleykowski (1998) Nucleic Acids Res.26：4597-4602；Qui etc. (2004) BioTechniques 36：702-707；Yeung etc. (2005) BioTechniques 38：749-758.

Show the result of two experiments for each sample in fig. 2.The experiment #2 of sample 8 and 9 has notable in road Background noise, which reduce apparent effect of these ZFN.

As shown in Fig. 2 some CCHC variants are substantially equal to wild type C2H2 ZFN.Refer to (the sample 5 of zinc finger 21 at 4 With 9) than referring to zinc finger 20 (sample 4 and 8) at 4 produce more preferable result.Referring to that zinc finger 43 produces best result in 2.

Embodiment 4：The intergenic suppression of eGFP in reporting cell line

Based on result shown in Fig. 1 and 2, CCHC zinc fingers (referred to as 1a to 10a) shown in table 3 above and 4 are generated.By these Zinc finger is mixed in 5-8 and 5-9ZFN, and is tested in GFP intergenic suppressions algoscopy described in example 2 above.Under each cylindricality Side shows ZFN pair tested in each sample, and wherein zinc finger numbering 20,21,43,45,47 and 48 is those in embodiment 3 Described in, and CCHC zinc fingers 1a to 10a is comprising sequence shown in table 3 above and 4.The zinc finger 20,21,7a used in referring to 4, 8a, 9a and 10a；The zinc finger 43,45,47,48,1a, 2a, 3a, 4a, 5a and 6a used in referring to 2.

Result is shown in figure 3.First trip below each cylindricality refers to the zinc finger mixed in ZFN 5-8, and each cylindricality The footline of lower section refers to the zinc finger mixed in ZFN 5-9.For example, the 2nd cylindricality on Fig. 3 from left side refers to 5-8 and 5-9ZFN The sample of transfection, the sequences of the F4 comprising zinc finger 20 of two of which ZFN.As shown, many includes the ZFN's of CCHC zinc fingers Performance is suitable with wild type (CCHH) ZFN.

Embodiment 5：The design and generation of targeting vector

A. the population structure of target sequence

Target construct for Nicotiana tabacum L. (dicotyledon) includes following 7 components, as shown in Figures 4 and 5：I) hygromycin phosphorus Sour transferring enzyme (HPT) expression cassette, it includes driving by Agrobacterium tumefaciens (A.tumifaciens) open reading-frame -24 (orf-24) Escherichia coli HPT genes that 3 ' untranslated regions (UTR) (Gelvin etc., 1987, EP222493) are terminated (Waldron etc., 1985, Plant Mol.Biol.18：Arabidopsiss (A.thaliana) ubiquitin -3 (ubi-3) promoter 189-200) (Callis etc., 1990, J.Bio.Chem.265：12486-12493)；Ii) homologous sequence -1, it includes Nicotiana tabacum L. (N.tabacum) RB7 matrix attachment regions (MAR) (Thompson etc., 1997, WO9727207)；Iii) 5 ' green fluorescent protein (GFP) genetic fragment (Evrogen Joint Stock Company, Moscow, Russia), it is by improveing Agrobacterium tumefaciens (Petolino etc., United States Patent (USP) No.6,730,824) are driven mannopine synthase (Δ mas) promoter；Iv) β-glucuronic acid Glycosidase (GUS) expression cassette, its include driving by Agrobacterium tumefaciens nopaline synthase (no) 3 ' UTR (DePicker etc., 1982, J.Mol.Appl.Genet.1：561-573) terminated gus gene (Jefferson, 1987, Plant Mol.Biol.Rep.5：Cassava vein mosaic virus (CsVMV) promoter 387-405) (Verdaguer etc., 1996, Plant Molecular Biology31：1129-1139)；V) by the UTR of Agrobacterium tumefaciens orf-1 3 ' (Huang etc., J.Bacteriol.172：1814-1822) terminated 3 ' GFP genetic fragments (Evrogen Joint Stock Company, Moscow, Russia)；Vi) homologous sequence -2, it includes arabidopsiss 4- coumaric acyl-CoA synthase (the 4-CoAS) (bases of intron -1 Because of seat At3g21320, GenBank NC 003074)；And vii) by the UTR (Gelvin of Agrobacterium tumefaciens ORF-25/26 3 ' Deng 1987, EP222493) transfer of green color-producing streptomycete (S.viridochromogenes) the phosphinothricin phosphoric acid that terminated Enzyme (PAT) (Wohlleben etc., 1988, Gene70：25-37) 3 ' genetic fragment.

By zinc finger-Fok1 fusion protein binding sites (IL-1-L0-Fok1) (Urnov etc., 2005, US2005/ 0064474) CsVMV promoteres (Verdaguer etc., 1996, Plant MolecularBiology 31 are inserted：1129-1139) Downstream, and with GUS coded sequences (Jefferson, 1987, Plant Mol.Biol.Rep.5：387-405) merge in N-terminal. 5 ' and 3 ' GFP genetic fragments (Evrogen Joint StockCompany, Moscow, Russia) flank has the second of two copies Zinc finger-Fok1 fusion protein binding sites (Scd27-L0-Fok1) (Urnov etc., 2005, US 2005/0064474).Each knot Close four tandem sequence repeats that specific zinc finger-Fok1 fusion protein recognition sequences are contained in site so that the size of each binding site For about 200bp (Fig. 6 A).This design is to guarantee that recognition sequence can be by zinc finger-Fok1 fusions in complicated chromatin environment Albumen institute is accessible.Each recognition sequence includes inverted repeat, and single zinc finger-Fok1 fusion protein is in homodimer form It is bound to the inverted repeat and cutting double-stranded DNA (Fig. 6 B).5 ' and 3 ' GFP genetic fragments are overlapping to reach 540bp, and this is provided Homology in target sequence, and termination codon is inserted in into 3 ' ends of 5 ' GFP fragments to guarantee not from the function of target sequence Property GFP translation.

Produce the conversion carrier comprising target sequence by multi-step cloning process as mentioned below.

The structure of B.HPT binary vectors (pDAB1584)

The fundamental construction body (Fig. 7) that carrier pDAB1400 containing GUS expression cassettes is used as to start, the GUS expression cassettes Comprising driving by Agrobacterium tumefaciens orf-1 UTR (Huang etc., J.Bacteriol.172：The GUS for 1814-1822) being terminated Gene (Jefferson, 1987, Plant Mol.Biol.Rep.5：Arabidopsiss ubi-3 promoter (Callis 387-405) Deng 1990, J.Bio.Chem.265：12486-12493).

Any unnecessary re-adjustments element, uses Agrobacterium tumefaciens orf-24UTR in order to eliminate target construct (Gelvin etc., 1987, EP222493) replace pDAB1400 in Agrobacterium tumefaciens orf-1UTR (Huang etc., J.Bacteriol.172：1814-1822), the Agrobacterium tumefaciens orf-24UTR is cut from as SacI/XbaI fragments PDAB782 (Fig. 8), and it is cloned into the same loci in pDAB1400.The construct of gained contains driving by Agrobacterium tumefaciens Gus gene that orf-24UTR (Gelvin etc., 1987, EP222493) is terminated (Jefferson, 1987, Plant Mol.Biol.Rep.5：Arabidopsiss ubi-3 promoteres 387-405) (Callis etc., 1990, J.Bio.Chem.265： 12486-12493), and it is named as pDAB1582 (Fig. 9).

Using primer P1 and P2 from pDAB354 plasmids (Figure 10) PCR amplification HPT coded sequences (Waldron etc., 1985, Plant Mol.Biol.18：189-200).In 5 ' the end addition BbsI sites of primer P1, and retain at the 3 ' ends of primer P2 SacI sites.HPTII PCR fragments are digested with BbsI/SacI, and are cloned in the pDAB1582 of Jing NcoI-SacI digestion, For replacing gus gene from the HPT genes of PCR fragment.The plasmid of gained is named as pDAB1583 (Figure 11).

Then arabidopsiss ubi-3/HPT/ Agrobacterium tumefaciens orf-24 pieces are cut out from pDAB1583 by NotI digestion Section, and it is polymerized ferment treatment to produce flat end with T4DNA.By the HPT expression cassettes processed through end-filling in PmeI sites gram It is grand enter pDAB2407 (Figure 12) (a kind of binary carrier is carrier), produce plasmid pDAB1584 (Figure 13).

C. the structure of the carrier (pDAB1580) of homologous sequence and Scd27 zinc finger-Fok1 fusion protein binding sites is included

Replaced in pDAB2418 (Figure 14) with Agrobacterium tumefaciens orf25/26UTR (Gelvin etc., 1987, EP222493) Agrobacterium tumefaciens orf-1UTR (Huang etc., J.Bacteriol.172：1814-1822), to eliminate targeting vector in weight Multiple regulatory sequence.In order to carry out UTP exchanges, using primer P3 and P4 from PCR amplifications crown gall soil in pDAB4045 plasmids (Figure 15) Earth bacillus orf25/26UTR (Gelvin etc., 1987, EP222493).In 3 ' end addition SmaI and AgeI sites of PCR fragment, And retain SacI sites at 5 ' ends.PDAB2418 plasmid DNA containing pat gene expression cassette is digested with SacI and AgeI, and Reclaim two kinds of maximums fragment, the pat gene expression cassette comprising drive by Agrobacterium tumefaciens orf-1UTR (Huang etc., J.Bacteriol.172：1814-1822) terminated pat gene (Wohlleben etc., 1988, Gene 70：Plan 25-37) Southern mustard ubiquitin -10 (ubi-10) promoter (Callis etc., 1990, J.Biol.Chem.265：12486-12493)) and Nicotiana tabacum L. RB7MAR sequences (Thompson etc., 1997, WO9727207).By these fragments and the crown gall soil of Jing SacI and AgeI digestion Bacillus orf25/26UTR (Gelvin etc., 1987, EP222493) PCR primer connects.The plasmid of gained is named as pDAB1575 (Figure 16).Nicotiana tabacum L. RB7MAR (Thompson etc., 1997, WO9727207) serves as the homologous sequence -1 in targeting vector.

The intron -1 (locus At3g21320, GenBank NC 003074) for selecting arabidopsiss 4-CoAS serves as target load Homologous sequence -2 in body.To pat gene (Wohlleben etc., 1988, Gene 70：25-37) coded sequence is analyzed, And will be accredited as the site for inserting intron at the 299/300bp of start codon downstream so that can be formed suitable 5 ' and 3 ' splice sites.Then by DNA synthesis (PicoscriptLtd., LLP, Houston, Texas) by total length intron with 3 ' the part PAT coded sequence fusions of 253bp.NotI and SacI sites are added to 5 ' and the 3 ' of DNA fragmentation is respectively held.Then The DNA fragmentation of synthesis is digested with NotI/SacI, and inserts pDAB1575 to replace total length PAT coded sequence in same loci. The construct of gained is named as pDAB1577 (Figure 17).

Synthesis (Picoscript Ltd., LLP, Houston, Texas) containing Scd27-L0-Fok1 recognition sites 4 The 241bp DNA fragmentations (Fig. 6) of tandem sequence repeats, wherein SmaI sites are added to 5 ' and 3 ' two ends of the fragment.Then will close Into the binding sites of-Fok1 containing zinc finger fragment with SmaI digest, and MscI sites insertion pDAB1577 in.The carrier of gained It is named as pDAB1579 (Figure 18).Then by the fragment of the binding sites of-Fok1 containing zinc finger of second Jing SmaI digestion in SwaI In site insertion pDAB1579.The construct of gained is named as pDAB1580 (Figure 19).The carrier comprising homologous sequence 1 and 2 (point Be not Nicotiana tabacum L. RB7MAR and arabidopsiss 4-CoAS introne 1s) and two synthesis Scd27 zinc finger-Fok1 binding sites (respectively contain 4 tandem sequence repeats of Scd27-L0-Fok1 recognition sites).

The structure of the carrier (pDAB1572) of the non-functional GFP fragments for D. repeating comprising two parts

GFP gene Cs opGFP are purchased from Evrogen Joint Stock Company (Moscow, Russia), and using drawing Thing P5 and P6 carry out PCR amplification complete encoding sequences.BbsI and SacI sites are added to 5 ' and the 3 ' of PCR primer is respectively held.So CopGFP PCR primers are digested with BbsI/SacI afterwards, and pDAB3401 (Figure 20) (its bag is cloned in NcoI/SacI sites Containing driving by Agrobacterium tumefaciens orf-1 3 ' UTR (Huang etc., J.Bacteriol.172：The GUS for 1814-1822) being terminated Gene (Jefferson, 1987, Plant Mol.Biol.Rep.5：Improvement Agrobacterium tumefaciens Δ mas 387-405) starts Sub (Petolino etc., US6730824)) replacing gus gene.The carrier of gained is named as pDAB1570 (Figure 21).

In order to prepare the non-functional GFP fragments that two kinds of parts are repeated, carry out PCR using primer P9 and P10 and expand following DNA Fragment, the DNA fragmentation contains the most of of CopGFP coded sequences but 5 ' ends have 47bp to delete.ApaI sites are added to 5 ' and 3 ' two ends, and extra StuI sites are added to 5 ' ends and in the downstream in ApaI sites.Then PCR primer is disappeared with ApaI Change, and pDAB1570 is inserted in ApaI sites, two NOT functions with 540bp repetitive sequences are thus produced in identical carrier Can property GFP fragments.The construct of gained is named as pDAB1572 (Figure 22).

E. the structure of the carrier (pDAB1573) of IL-1 zinc finger-Fok1 fusion protein binding sites/gus gene fusions is included Build

By Picoscript Ltd., LLP, (Houston, Texas) synthesis comprising IL-1_L0-Fok1 recognition sites 4 The 233bp DNA fragmentations (Fig. 6) of individual tandem sequence repeats, wherein respectively adding NcoI and AflIII sites to 5 ' and 3 ' ends.Then The fragment of synthesis is digested with NcoI/AflIII, and in NcoI sites insertion pDAB4003 (Figure 23), it includes and is opened by CsVMV Mover (Verdaguer etc., 1996, PlantMolecular Biology 31：1129-1139) drive, by crown gall soil bar Bacterium orf-1 3 ' UTR (Huang etc., J.Bacteriol.172：1814-1822) terminated gus gene (Jefferson, 1987, Plant Mol.Biol.Rep.5：387-405).Then IL-1_L0-Fok1 binding sites and GUS code sequences are generated N-terminal fusions between row.The carrier of gained is named as pDAB1571 (Figure 24).

The UTR elements of repetition 3 ' in order to eliminate targeting vector, by the UTR of Agrobacterium tumefaciens no 3 ' (DePicker etc., 1982, J.Mol.Appl.Genet.1：561-573) cut out from pDAB7204 (Figure 25) as SacI/PmeI fragments, and gram It is grand enter Jing SacI/NaeI digestion pDAB1571 with replace the UTR of Agrobacterium tumefaciens orf-1 3 ' (Huang etc., J.Bacteriol.172：1814-1822).The plasmid of gained is named as pDAB1573 (Figure 26).

F. the structure of final targeting vector (pDAB1585)

In order to build final targeting vector, cut out from pDAB1573 by NotI digestion and merge egg with IL-1-Fok1 The GUS expression cassettes of white target site insertion, filling-in end, and insert pDAB1572 in StuI sites.The intermediate carrier name of gained For pDAB1574 (Figure 27).Cut out complete box from pDAB1574, and pDAB1580 is inserted in NotI sites, it is described complete Box repeats GFP sequences (Evrogen Joint comprising improvement Δ mas promoteres (Petolino etc., US6730824), 5 ' parts StockCompany, Moscow, Russia), CsVMV promoteres (Verdaguer etc., 1996, PlantMolecular Biology 31：1129-1139), IL-1-Fok1 fusion protein target sequence, gus gene (Jefferson, 1987, Plant Mol.Biol.Rep.5：387-405) coding region, the UTR of Agrobacterium tumefaciens no 3 ' (DePicker etc., 1982, J.Mol.Appl.Genet.1：561-573), 3 ' part repeat GFP (Evrogen Joint Stock Company, Moscow, ) and Agrobacterium tumefaciens orf-1 3 ' UTR (Huang etc., J.Bacteriol.172 Russia：1814-1822).The matter of gained Grain is named as pDAB1581 (Figure 28).Then the AgeI fragments of pDAB1581 are inserted into pDAB1584 in AgeI sites, is thus produced Final target construct pDAB1585 (Figure 4 and 5) of life.

Embodiment 6：The generation of the transgenic cell line with the target sequence integrated

Using tobacco cell suspension culture (it is referred to as BY2), the stable integration thereto via Agrobacterium conversion The target sequence of embodiment 5.Basal cell system BY2 available from Jun Ueki (Japan Tobacco, Iwata, Shizuoka, Japan).Culture propagation in 100-150 cell cluster (cell cluster) is the cell of 5-10 μ diameters, wherein times The increasing time is substantially 18 hours.BY2 cell suspension cultures are maintained in the culture medium containing following ingredients：LS bases salt (PhytoTechnology Labs L689)、170mg/L KH₂PO₄, 30g/L sucrose, 0.2mg/L 2,4-D and 0.6mg/L salt Allithiamine element, pH6.0.By add 50mL based on the culture medium (LS-based medium) of LS to 0.25mL PCV come per 7 days Secondary Culture is carried out to BY2 cells.BY2 cell suspension cultures are maintained on rotary shaker (25 DEG C, 125RPM) In 250mL flasks.

In order to produce the transgenic BY2 cell cultures with the target sequence integrated, by the cigarette of 4 days after one bottle of Secondary Culture Careless suspension is divided into the aliquot of 10-12 part 4mL, by itself and 100 μ L incubated overnight to OD₆₀₀About 1.5 comprising pDAB1585 Edaphic bacilluss bacterial strain LBA4404 co-culture in 100x25mm Pi Shi wares.Each ware is wrapped up with Parafilm, and at 25 DEG C Incubate in the case where not shaken 3 days, afterwards, excessive liquid is removed, and contain 500mg/L carboxylic benzyl penicillium sp with 11mL The basal medium based on LS of element is replaced.

After making tobacco cell resuspension, by 1mL suspensions distribute to 8g/L TC it is agar solidified, containing 500mg/L carboxylics On the 100x25mm flat boards of the appropriate basal medium of benzylpcnicillin and 200mg/L hygromycin, and at 28 DEG C in the form of not wrapping up Incubate in the dark.This causes single treatment to produce 120-144 blocks selection flat board.There is an other tide within 10-14 days after bed board Chloramphenicol resistance separates group (isolate), and transfers them to each 60x20mm flat board (one separation group of every piece of flat board), wherein According to 14 days Secondary Culture schedules by them with the maintenance of corpus callosum form, until needing to convert reality again with subsequent for analysis Test.

Embodiment 7：The screening of target transgenic event and sign

To the hygromycin resistance transgenic event produced by BY2 tobacco cell cultures is transformed into from targeting vector (as implemented Described in example 6) analyzed as follows.

What the initial analysis carried out to screen these transgenic events included GUS expression analysis to show target sequence can The PCR of proximity, part and total length target sequence analyzes to confirm presence and integrity and the Southern engram analysis of targeting vector To determine the copy number of integrated target sequence.Show a subset of transgenic event of GUS expression comprising a single copy Total length target sequence；Target system for rebuilding suspension culture to produce for subsequent conversion again is selected to them.These are heavy The target system for building is also subject to further sign, and it includes more thorough Southern engram analysis, the sequencing of whole target insert Confirmation and flanking genomic sequence analysis.

It is active that GUS is analyzed the transgene tobacco corpus callosum that starts from selected event or suspension culture as follows, will 50mg samples incubate 24-48 hours in 150 μ L determine buffer at 37 DEG C.Measure buffer sodium phosphate containing 0.2M pH 8.0, The potassium ferricyanide and potassium ferrocyanide of each 0.1mM, the chloro- 3- indyls-β-Portugals of the bromo- 4- of 1.0mM sodium salt EDTA, 0.5mg/mL 5- Glycuronide (glucuronide) and 0.6% (v/v) Triton X-100 (Jefferson, 1987, Plant Mol.Biol.Rep.5：387-405).By the indicant of the appearance as gus gene expression of blue colored areas, it shows target It, with transcriptional activity, therefore is come-at-able in the genomic context of local that sequence insertion is.

The transgenic event for expressing GUS is measured by using the PCR of primer pair P15/P16, described use is drawn Thing causes from 3 ' UTR of the HTP expression cassettes at the end of target sequence 5 ' to extend to the part PAT bases at the end of target sequence 3 ' to the PCR of P15/P16 Because of the 10kb DNA fragmentations amplification of 3 ' UTR of box.Because all of event is obtained in the case where hygromycin is selected, it is taken as that HPT expression cassettes are all complete in all target events.Therefore, 3 ' UTR of HPT expression cassettes are only covered in the analysis of total length PCR.Also Performing PCR measure is entered to determine the 5 ' and 3 ' of target sequence respectively using primer pair P15/P17 and P18/P19 to a subset of event The integrity at end.All Jing PCR are analyzed with the target event for confirming by Southern engram analysis carries out further determining to survey The copy number of fixed integrated target sequence.

To all target event implementation Southern engram analysis screened by GUS expression and total length PCR.By 10 μ g genes A cleaver (unique cutter) NsiI digestion in group DNA target sequences.The genomic DNA that Jing is digested is 0.8% Separate on agarose gel, and be transferred on nylon membrane.After cross-linking, the transfer on the film is hybridized with HPT gene probes DNA with determine target sequence 5 ' end copy numbers.Then identical trace is peeled off into (strip), and it is miscellaneous again with pat gene probe Hand over to determine the copy number at 3 ' ends of target sequence.

Multiple events for showing that GUS is expressed and copied total length target sequence containing list are selected to be used to further characterize, its bag Include more thorough Southern engram analysis, whole target sequence confirmation and flanking genomic sequence analysis.Selected based on characterization of molecules Select an event for being referred to as BY2-380.From the event reconstruction suspension culture, for load of the subsequent use comprising donor dna What body and non-C2H2 zinc fingers-Fok1 antigen-4 fusion protein genes were carried out converts again.

In order to ensure the suspension culture set up from target event BY2-380 is comprising expected complete target sequence, using primer Target sequence main body is expanded to P15/P16PCR (from the part that 3 ' UTR of the HPT expression cassettes at the end of target sequence 5 ' hold to target sequence 3 ' 3 ' UTR of pat gene box), and it is cloned into pCR2.1TOPO carriers (Invitrogen, Carlsbad, California).TOPO By Larktechnology, Inc. (Houston, Texas) is sequenced the PCR primer inserted in carrier.Sequence results indicate, BY2-380 has expected complete target sequence.

Come to BY2- using general GenomeWalker test kits (Clontech, Mountain View, California) 380 cell lines are further analyzed to obtain flanking genomic sequence.In short, three kinds of flush ends used in separate reaction Restriction enzyme EcoRV, DraI and StuI are digesting 2.5 μ g genomic DNAs.The DNA that Jing is digested is by phenol/chloroform come pure Change, and be connected with BD GenomeWalker adapters.Implement nested PCR amplification, the wherein connection as template and primer P20 (in 5 ' end upstream walkings of target sequence insertion) and P21 (in 3 ' end downstream walkings of target sequence insertion) are anti-for one-level PCR Should, and primer P22 (in 5 ' end upstream walkings of target sequence insertion) and P23 (in 3 ' end downstream walkings of target sequence insertion) are used for Two grades of nested PCR reactions.PCR2.1TOPO or pCR Blunt II will be cloned into from the amplified fragments of two grades of PCR reactions TOPO carriers (Invitrogen, Carlsbad, California), and using Dye Terminator thing cycle sequencing test kit (Dye Terminator Cycle Sequencing Kit, Beckman Coulter, Fullerton, CA) being sequenced.Via the party Method obtains flanking genomic sequence from BY2-380 targets system.Flanking genomic sequence design primer is then based on, and for expanding Whole target sequence.

The amplified fragments obtained from the target system have expected size.Two ends of amplified fragments are confirmed by sequencing.

Embodiment 8：The design and generation of donor dna carrier

Donor DNA constructs include homologous sequence -1 (Nicotiana tabacum L. RB7MAR) (Thompson etc., 1997, WO9727207), complete Long arabidopsiss ubi10 promoteres (Callis etc., 1990, J.Biol.Chem.265：12486-12493), the 5 ' parts of 299bp Pat gene coded sequence (Wohlleben etc., 1988, Gene70：25-37) and homologous sequence -2 (arabidopsiss 4-CoAS is included Son -1) (locus At3g21320, GenBank NC 003074).Homologous sequence -1 and sequence -2 in donor vehicle both with Corresponding homologous sequence -1 and sequence -2 are identicals in targeting vector (pDAB1585).

In order to build donor vehicle, by Picoscript Ltd., LLP, the DNA synthesis that (Houston, Texas) is carried out By 5 ' the part PAT coded sequences of 299bp and total length arabidopsiss 4-CoAS introns -1 (locus At3g21320, GenBank NC 003074) fusion.NcoI and XhoI sites are added to 5 ' and the 3 ' of the fragment is respectively held.Then this is closed Into DNA fragmentation digested with NcoI/XhoI, and insert pDAB1575 replacing total length pat gene coded sequence in same loci And its 3 ' UTR.The construct of gained is named as pDAB1576 (Figure 29).

Then pDAB1576 is digested with AgeI, and by comprising the 5 ' parts that flank is homologous sequence -1 and homologous sequence -2 Pat table inserts pDAB2407 (a kind of binary carrier is carrier) up to the whole fragment of box in same loci.The construct name of gained For pDAB1600 (Figure 30), and it is the donor vehicle for the plant cell binary pattern of conversion again.

Embodiment 9：The design and generation of Zinc finger nuclease expression vector

Zinc finger-Fok1 antigen-4 fusion protein genes driven by CsVMV promoteres and 5 ' UTR (Verdaguer etc., 1996, Plant Molecular Biology 31：1129-1139).What is also included in the box is Agrobacterium tumefaciens open reading-frame -24 (orf-24) 3 ' untranslated regions (UTR) (Gelvin etc., 1987, EP222493).

In order to build these carriers, IL-1-Fok1 and Scd27-Fok1 coded sequences described in example 1 above to 4 C2H2 compare and its C3H variants expand from PCR in their initial design, respectively by BbsI or NcoI and SacI sites add to The 5 ' of PCR fragment and 3 ' ends, and be cloned in the pDAB3731 (Figure 31) of Jing NcoI-SacI digestion.The plasmid of gained is named as PDAB4322 (Figure 32), pDAB4331 (Figure 33), pDAB4332 (Figure 34), pDAB4333 (Figure 35), pDAB4334 (Figure 36), PDAB4336 (Figure 37) and pDAB4339 (Figure 38).AttL1 and attL2 position of all these carriers comprising ZFN expression cassette flanks Point, and and Gateway^TMCloning system (Invitrogen, Carlsbad, California) is compatible.

For two sets of binary pattern carriers of IL-1-FokI fusion protein constructions.It is a set of comprising PAT selected marker genes, it is and another It is a set of not comprising PAT selected marker genes.For SCd27-FokI fusion protein, only build without PAT selected marker genes Binary pattern carrier.In order to build the binary vector containing PAT selected marker genes, using LR Clonase^TMEnzymatic mixture (Invitrogen, Carlsbad, California) via LR recombining reactions by pDAB4322, pDAB4331, pDAB4332, IL-1-FokI expressing fusion protein boxes in pDAB4333, pDAB4334 and pDAB4336 are cloned into pDAB4321 (Figure 39) In.The plasmid of gained is named as pDAB4323 (Figure 40), pDAB4341 (Figure 41), pDAB4342 (Figure 42), pDAB4343 (figures 43), pDAB4344 (Figure 44), pDAB4346 (Figure 45).In order to build the binary vector without PAT selected marker genes, use LR Clonase^TMEnzymatic mixture (Invitrogen, Carlsbad, California) will exist respectively respectively via LR recombining reactions C2H2IL-1-FokI, C3H IL-1-FokI and Scd27-FokI expression cassettes in pDAB4331, pDAB4336 and pDAB4339 In being cloned into pDAB4330 (Figure 46).Gained plasmid be respectively designated as pDAB4351 (Figure 47), pDAB4356 (Figure 48) and PDAB4359 (Figure 49).

In order to the C2H2 for building SCD27-FokI is compareed, with comprising the CsVMV promoteres and 5 ' UTR and Nicotiana tabacum L. for driving GUS Bag in fragment (its cutting from pDAB7025 (Figure 51) with HindIII/SacI) the replacement pDAB7002 (Figure 50) of 5 ' UTR HindIII/SacI fragments containing the CsVMV promoteres and 5 ' UTR for driving PAT.The plasmid of gained is named as pDAB1591 (figures 52).Scd27-L0-Fok1 coded sequences use primer pair P13/P14 from its initial carrier pCDNA3.1-SCD27a-L0-FokI (Figure 53) PCR amplifications.BbsI and SacI sites are added to 5 ' and the 3 ' of PCR fragment is respectively held.Use via SacI/NcoI clones Zinc finger antigen-4 fusion protein gene PCR fragment replaces the pat gene in pDAB1591.The plasmid of gained is named as pDAB1594 (figures 54).The binary pattern of the carrier is constructed as below, i.e., zinc finger fusion protein base is cut out with PmeI/XhoI fragments from pDAB1594 Because of expression cassette, filling-in end, and pDAB2407 is cloned in PmeI sites.The plasmid of gained is named as pDAB1598 (Figure 55). The details of all binary vectors for Plant Transformation are summarized in table 6.

Table 6：Zinc finger nuclease expression vector

FokI domains are plant codon-bias.

Embodiment 10：The design and generation of positive control vector

In order to assess unconventional recombination frequency and serve as positive control, the carrier containing pat gene expression cassette is used.In order to Can be compared with final recombinant, PAT coded sequences (Wohlleben etc., 1988, Gene 70：299/ 25-37) Arabidopsiss 4-CoAS introns -1 (locus At3g21320, GenBank NC 003074) is inserted at 300bp.In order to build this Construct is planted, the identical pDAB1577 (figures for limiting enzymic digestion of Jing will be connected from the 2559bpSwaI/ClaI fragments of pDAB1576 56) backbone segments.Gained carrier comprising pat gene expression cassette and its PAT coded sequences (Wohlleben etc., 1988, Gene70：There is (locus At3g21320, the GenBank NC of arabidopsiss 4-CoAS introns -1 of 1743bp in the middle of 25-37) 003074) (locus At3g21320, GenBank NC 003074) insertion.The carrier is named as pDAB1578 (Figure 57).

In order to build the binary pattern of pDAB1578, cut out from pDAB 1578 with PmeI/XhoI and included with arabidopsiss The pat gene expression cassette of -1 (locus At3g21320, GenBank NC 003074) of son.Mend at the 3 ' ends to the fragment After flat end-o f-pipe -control, it is inserted into binary carrier is carrier pDAB2407 in PmeI sites.The carrier of gained is named as pDAB1601 (Figure 58), its include by arabidopsiss ubi10 promoteres (Callis etc., 1990, J.Biol.Chem.265：12486-12493) institute Drive and by Agrobacterium tumefaciens orf25/263 ' UTR (Gelvin etc., 1987, EP222493) terminated comprising arabidopsiss 4-CoAS introns -1 (locus At3g21320, GenBank NC 003074) pat gene (Wohlleben etc., 1988, Gene 70：25-37).

Embodiment 11：By the way that with C3H Zinc finger nucleases gene, again transformed target cell culture is intrachromosomal same to prove Recombinate in source

In order to confirm feature of the C3H Zinc finger nucleases in Intrachromosomal recombination is stimulated, as shown in figure 59, target is carried Body includes with 540bp overlapping sequences two non-functional GFP fragments.Between the two fragments is gus gene table Up to box.By IL-1-Fok1 fusion protein binding sequence and GUS coded sequences in its N-terminal fusion.It is not limited to a kind of theory, it is assumed that In the presence of IL-1-Fok1 fusion protein, IL-1ZFN binding sequences can be identified, and double-strand DNA cleavage can be induced, this Interior source DNA repair process can be stimulated.In the presence of without donor dna, the GFP fragments of two homeologouses can experience dyeing in vivo Homologous recombination process, and feature GFP gene can be reconstructed.

It is again started up hanging using BY2-380 transgenic cell lines (it contains single copy, the target sequence that total length is integrated) Floating culture, will about 250-500mg callus tissues be put into the basis training based on LS that 40-50mL contains 100mg/L hygromycin In foster base, and per 7 days carried out Secondary Culture as above.Before converting again, suspension culture is transferred to and is not contained The basal medium of hygromycin reaches at least two generations.

Implement agrobacterium-mediated target cell culture conversion as described above.It is following to produce 8 pieces for each experiment Co-culture flat board：1 piece of flat board includes the cell co-cultured with 300 μ L foundation soil bacillus strains LBA4404；1 piece of flat board is included The cell co-cultured with edaphic bacilluss bacterial strains of the 300 μ L comprising pDAB1590 (feature GFP construct)；6 pieces of flat boards are respectively included With the soil that 300 μ L include respectively pDAB4323, pDAB4341, pDAB4342, pDAB4343, pDAB4344 and pDAB4346 The cell that bacillus strain is co-cultured.After being co-cultured using method as described above, cell is laid on equipped with being supplemented with 500mg/L Carbenicillins and do not contain on 8 pieces of flat boards of the basal medium based on LS of selective agent.The feature set up The apparent expression of GFP genes causes about 5-8 days visible fluorescence after conversion.It is micro- by observing every piece of flat board 5 " random " The mirror visual field is carried out the number of the green fluorescence position to each visual field and is counted (8 pieces of flat boards of every kind of construct in each experiment), and Meansigma methodss are calculated from 6 independent experiments.

As table 7 is summarized, observe that each is regarded respectively from two kinds of C3H Zinc finger nucleases, i.e. pDAB4346 and pDAB4343 Wild average 9.50 and 7.57 green fluorescence positions.Both C3H designs of IL-1-FokI are compareed than its C2H2, i.e., PDAB4341 (6.37, each visual field position) and pDAB4323 (5.53, each visual field position) performance are more preferable.Meanwhile, with C2H2 controls are compared, the other two C3H variant of IL-1-FokI fusion protein be pDAB4344 (4.39, each visual field position) and The function of pDAB4342 (0.25, each visual field position) is significantly damaged, and particularly pDAB4342, wherein C3H conversion only passes through Replacing second cysteine with histidine in referring at the 4th is carried out.In the moon converted with foundation soil bacillus strain LBA4404 Property control in the fluorescence for perceiving more than slight background is not observed.

Table 7

The structure of feature GFP that the Intrachromosomal recombination stimulated via IL-1-Fok1 zinc fingers fusion protein is carried out

Carrier	ZFP types	GFP is expressed	Tukey is checked**
				pDAB4346	C3H	9.50	A
pDAB4343	C3H	7.57	B
				pDAB4341	C2H2	6.37	C
pDAB4323*	C2H2	5.53	D
				pDAB4344	C3H	4.39	E
pDAB4342	C3H	0.25	F

^*FokI domains comprising non-plant codon-bias

^**The average not contacted with same letter has significant difference in 0.05 level

Embodiment 12：By with C3H Zinc finger nucleases gene and donor DNA sequences again transformed target cell culture proving Interchromosomal homologous recombination

In order to confirm C3H zinc finger-Fok1 fusion protein in exemplary Tobacco System is stimulated in interchromosomal homologous recombination Feature, develop and test two kinds of strategies.

In strategy 1, the middle part of target construct (Figure 61) includes zinc finger-Fok1 fusion protein binding site (IL-1-L0- Fok1).In the strategy, binding site both sides flank is for about the nonhomologous sequence of 3kb, and then upstream and downstream is respectively same Source sequence -1 (Nicotiana tabacum L. RB7MAR) and homologous sequence -2 (arabidopsiss 4-CoAS introns -1).As previously confirmed (such as U.S. Patent Publication No.20050064474), in the presence of C2H2IL-1 zinc finger-Fok1 fusion protein, IL-1-L0-Fok1 knots Close sequence to be identified, and in the specific site induction double-strand DNA cleavage, this stimulates interior source DNA repair process.In donor dna In the presence of (it is included and the homologous sequence identical homologous sequence in target sequence), 5 ' part pat genes and its promoter via Homologous recombination to replace target in whole about 6kb DNA fragmentations between homologous sequence.By this method, two part PAT bases Because of sequence (arabidopsiss 4-CoAS introns -1 interleave therebetween) Reconstruction of The Function pat gene, pat table is caused to reach and Herbicid resistant Phenotype.

In strategy 2, targeting vector includes two zinc fingers-Fok1 binding sites (Scd27-L0-FokI)：One is placed exactly in Nicotiana tabacum L. RB7MAR downstreams, and another is placed exactly in the upstream (Figure 62) of arabidopsiss 4-CoAS introns -1.Two zinc finger-Fok1 melt Between hop protein binding site is the sequence of about 6kb, and it includes 5 ' GFP fragments, GUS expression cassettes and 3 ' GFP fragments.As previously Confirm (such as United States Patent (USP) disclosure No.20050064474), in the presence of Scd27 zinc finger-Fok1 fusion protein, two Individual binding sequence is identified, and in two position induction double-strand DNA cleavages, this removes the pact between the two binding sequences 6kb DNA fragmentations, and source DNA repair process in stimulating.It is 1 similar with strategy, donor dna (its include with it is same in target sequence Source sequence identical homologous sequence) in the presence of, 5 ' part pat genes and its promoter are by the position of induction double-strand DNA cleavage The homologous recombination that carries out at point and be inserted into target sequence.By this method, two part pat gene sequence (arabidopsiss 4- CoAS introns -1 interleave therebetween) Reconstruction of The Function pat gene, cause pat table to reach and herbicide resistant phenotype.

Implement agrobacterium-mediated BY2-380 target cell cultures conversion as described above.It is as follows for each experiment Produce 12 pieces of co-cultivation flat boards：1 piece of flat board includes the edaphic bacilluss bacterial strain and 250 μ that pDAB1600 (donor dna) is included with 50 μ L The cell that L edaphic bacilluss basis bacterial strain LBA4404 is co-cultured；1 piece of flat board is included with 50 μ L comprising pDAB1601 (PAT selection marks Will) edaphic bacilluss bacterial strain and the cells that co-culture of 250 μ L edaphic bacilluss basis bacterial strain LBA4404；2 pieces of flat boards are included and 50 μ L The soil of edaphic bacilluss bacterial strain comprising pDAB1600 (donor dna) and 250 μ L comprising pDAB4351 (C2H2IL-1ZFP-Fok1) The cell that earth bacillus strain is co-cultured；3 pieces of flat boards include with 50 μ L comprising pDAB1600 (donor dna) edaphic bacilluss bacterial strains and The cell that edaphic bacilluss bacterial strains of the 250 μ L comprising pDAB4356 (C3H IL-1ZFP-Fok1) is co-cultured；2 pieces of flat boards are included and 50 Edaphic bacilluss bacterial strains of the μ L comprising pDAB1600 (donor dna) and 250 μ L include pDAB1598 (C2H2Scd27a ZFP-Fok1) Edaphic bacilluss bacterial strain co-culture cell；3 pieces of flat boards include edaphic bacilluss bacterial strains of the 50 μ L comprising pDAB1600 (donor dna) The cell co-cultured with edaphic bacilluss bacterial strains of the 250 μ L comprising pDAB4359 (C3H Scd27aZFP-Fok1).Using institute above After the method for stating is co-cultured, cell is spread into containing 500mg/L Carbenicillins and 15mg/LBased on LS Basal medium on.2-4 weeks occur individual other after bed boardResistant segregation group, and transfer them to each 60x20mm flat boards (every piece flat board one separation group), wherein according to 14 days Secondary Culture schedules by them in corpus callosum form Maintain, until needing for analyzing.

All obtain many from C3H IL-1 Zinc finger nucleases (pDAB4356) and C3H Scd27 Zinc finger nucleases (pDAB4359) It is individualResistant segregation group.By using the PCR of primer pair P24/25, (its amplification is across the pat gene rebuild DNA fragmentation) come analyze these separate group.5 ' ends of PAT coded sequences are homologous, and primers in primer P24 and donor dna P25 is homologous with 3 ' ends of PAT coded sequences in target DNA.If two part PAT coded sequences are connected by homologous recombination Connect, then can produce 2.3kbPCR fragments.As shown in Figure 63,2.3kb PCR primers are obtained from the multiple separation groups for being analyzed.From C3H IL-1 zinc fingers-Fok1 antigen-4 fusion protein genes/donor dna and C3H Scd27 zinc fingers-Fok1 antigen-4 fusion protein genes/donor dna Cotransformation in all obtain these separate group.Will from multiple independent separate groups (its represent from C3H IL-1 zinc finger-Fok1 and The conversion of C3H Scd27 zinc finger-Fok1 antigen-4 fusion protein genes both derivative separate groups) 2.3kb PCR primers it is solidifying from agarose Purification in glue, and it is cloned into pCR2.1TOPO carriers (Invitrogen, Carlsbad, California).Then dyestuff is used Terminate thing cycle sequencing test kit (Beckman Coulter) the 2.3kb PCR primers inserted in TOPO carriers are sequenced. Sequencing result confirms that all PCR primers cloned in TOPO carriers include the recombination sequence predicted, it includes 5 ' and 3 ' parts Pat gene sequence and the arabidopsiss 4-CoAS introns -1 for interleaving.These results are confirmed and merged via C3H zinc fingers-Fok1 Protein gene expression there occurs the interchromosomal recombination of two kinds of strategy predictions to being tested and illustrated gene target.

Embodiment 13：The identification of corn cell culture target gene sequence

A. Sequence Identification

In the present embodiment, the DNA sequence of endogenous corn gene of known function is selected as using through engineering approaches zinc finger core The target of the genome editor that sour enzyme is carried out.The gene (referred to as IPP2-K, its Semen Maydiss inbred line 5XH751 derived from sale monopoly) Genome structure and sequence be recorded in WO2006/029296, the disclosure of which is included by referring to.

Especially, using BLAST algorithm, TIGR Maize genome data bases are inquired about using IPP2-K genome sequences (can on the internet in http://www.tigr.org/tdb/tgi/maize/ is obtained).Identify several other gene pack Section has the section for having overlapping homology with IPP2-K, including but not limited to accession number AZM515213 and TC311535.Based on this The sequence and IPP2-K sequences of a little accession number, using Primer3 programs (Rozen, S. and Skaletsky, H.J. (2000) Primer3 on the WWW forgeneral users and for biologist programmers. in：Krawetz S, Misener S (eds.) Bioinformatics Methods and Protocols：Methods in Molecular Biology.HumanaPress, Totowa, NJ, pp 365-386；Also can obtain on the internet), design multiple short few cores Thuja acid, for use as PCR primer.These primers include but is not limited to following positive oligonucleotide：

1.5’-ATGGAGATGGATGGGGTTCTGCAAGCCGC-3’(SEQ ID NO：104)

2.5’-CTTGGCAAGGTACTGCGGCTCAAGAAGATTC-3’(SEQ ID NO：161)

3.5’-ATGAAGAAAGACAGGGAATGAAGGAC-3’(SEQ ID NO：162)

4.5’-ATGAAGAAAGACAGGGAATGAAGGACCGCCAC-3’(SEQ ID NO：163)

5.5’-CATGGAGGGCGACGAGCCGGTGTAGCTG-3’(SEQ ID NO：164)

6.5’-ATCGACATGATTGGCACCCAGGTGTTG-3’(SEQ ID NO：165)

In addition, primer includes but is not limited to following reverse oligonucleotide：

7.5’-TTTCGACAAGCTCCAGAAAATCCCTAGAAAC-3’(SEQ ID NO：166)

8.5’-ACAAGCTCCAGAAAATCCCTAGAAACAC-3’(SEQ ID NO：167)

9.5’-TTCGACAAGCTCCAGAAAATCCCTAGAAACAC-3’(SEQ ID NO：168)

10.5’-TGCTAAGAACATTCTTTTCGACAAGCTCC-3’(SEQ ID NO：169)

11.5’-GAACATTCTTTTCGACAAGCTCCAGAAAATCC-3’(SEQ ID NO：170)

All oligonucleotide primers are closed by Integrated DNA Technologies (IDT, Coralville, IA) Into and from its purchase.

B.Hi II corn cell cultures

In order to obtain the immature embryo for starting corpus callosum culture, in Hi-II the parent A and B of hot-house culture (Armstrong, C., Green, C. and Phillips, R. (1991) Maize Genet.Coop.News Lett.65：92-93) Between carry out F₁Hybridization.The embryo of size about 1.0-1.2mm (about 9-10 days after pollination) is harvested from the fringe of health, and by use Liqui-Soap is cleaned to carry out surface sterilizing, the submergence 2-3 minutes in 70% ethanol, then floats in 20% commercialization Submergence 30 minutes in white agent (0.1% sodium hypochlorite).

Fringe is rinsed in aseptic distilled water, and aseptically cuts out immature zygotic embryo, and in 15Ag10 trainings Foster base (N6 culture medium (Chu C.C., Wang C.C., Sun C.S., Hsu C., Yin K.C., Chu C.Y., and Bi F.Y. (1975)Sci.Sinica 18：659-668), 1.0mg/L 2,4-D, 20g/L sucrose, 100mg/L casein hydrolysate (enzymatics Digest), 25mM L-PROLINEs, 10mg/L AgNO₃, 2.5g/L Gelrite, pH5.8) on cultivate 2-3 it is all, its dorsolum (scutellum) dorsad culture medium.By show desired morphology tissue (Welter, ME, Clayton, DS, Miller, MA, Petolino, JF. (1995) Plant Cell Rep：14：725-729) it is selectively transferred to interval biweekly fresh In 15Ag10 culture medium, up to about 6 weeks, but 4 culture medium (N6 culture medium, 1.0mg/L2,4- are transferred to interval biweekly D, 20g/L sucrose, 100mg/L casein hydrolysates (enzymatic digestion thing), 6mM L-PROLINEs, 2.5g/L Gelrite, PH5.8 on), up to about 2 months.

In order to start embryo occur suspension culture, will about 3ml enrich cell volume (PCV) the corpus callosum from single embryo Tissue adds to about 30ml H9CP+ fluid mediums (MS bases salt mixture (Murashige T. and Skoog F. (1962)Physiol.Plant.15：473-497), the improvement MS dimensions containing few 10 times nicotinic acid and high 5 times of thiamine hydrochloride Raw element, 2.0mg/L 2,4-D, 2.0mg/L α-naphthaleneacetic acid (NAA), 30g/L sucrose, (the acid digestion of 200mg/L casein hydrolysates Thing), 100mg/L muscle-inositol, 6mM L-PROLINEs, 5%v/v Sucus Cocoiss (just before Secondary Culture add), pH6.0). Maintain to suspend in 125ml Erlenmeyer flasks in 125rpm, 28 DEG C of temperature control bottle swingging machine is arranged under dark condition and train Foster thing.During Establishment of Cell Line (2-3 month), by using macropore pipet by 3ml PCV cells and 7ml conditioning cultures Base adds to the fresh H9CP+ fluid mediums of 20ml to carry out Secondary Culture to suspension per 3.5 days.Reach maturation (such as to pass through Growth multiplication is proved) after, suspension is amplified in 500ml flasks, and maintain, thus by 12ml PCV cells and 28ml Conditioned culture media is transferred in 80ml H9CP+ culture medium.After suspension culture is completely set up, aliquot is carried out low Warm preservation, for future usage.Referring to WO 2005/107437.

C.DNA is separated and expanded

In standard GN6 culture medium (N6 culture medium, 2.0mg/L 2,4-D, 30g/L sucrose, 2.5g/L in 250ml flasks Gelrite, pH5.8) in culture Semen Maydiss HiII cell cultures mentioned above, and using Qiagen (Valencia, CA) plant DNeasy extracts kits extract genomic DNA according to the recommendation of manufacturer.Implement under the following conditions with all possible group Close using the pcr amplification reaction of primer mentioned above：25 μ l reaction volumes, in the buffer of enzyme manufacturer 20ng is contained The Accuprime of the every kind of primer of gDNA templates, 20pmol, 1% DMSO and 10 unit^TMPf polymerases (Invitrogen, Carlsbad, CA).The amplified production of magnitude range 500bp to 2kb be derived from by 95 DEG C -1 ', (95 DEG C -30 ", 57-62 DEG C -30 ", 72 DEG C -1 ') X30,72 DEG C -5 ', the amplification cycles of 4 DEG C-holding composition.Using TA Cloning Kits (Invitrogen, Carlsbad, CA) according to manufacturer recommendation by amplified fragments Direct Cloning enter carrier pCR2.1 (Invitrogen, Carlsbad, CA) in.

D. sequence analysis

Composition has been indicated to the previous analysis of IPP2-K genes in Semen Maydiss inbred line 5XH751 and HiII cell culture The 2-3 of mini-gene family heterogeneic presence (Sun etc., in printing, PlantPhysiology；WO2006029296).Cause This, with CEQ Dye Terminator thing cycle sequencing test kits (CEQDye Terminator Cycle Sequencing Kit, Beckman Coulter, Fullerton, CA) separated cloned sequence is sequenced according to the recommendation of manufacturer.To multiple grams Grand sequence analysis are disclosed, and 2 different genetic fragments are isolated from HiII cells, and it is derived from Maize genome 2 locus that are different and previously having characterized.

From relatively indicating for detached 2 sequences of HiII cultured cells, in predictive coding area, 2 collateral line homologue it Between there is small difference (such as single nucleotide polymorphism, SNP), and intron and noncoding region in nucleotide level significantly not Together.Notice that these differences between 2 collateral line homologues are because that they are highlighted and can combine egg by sequence dependent DNA The sequence area that in vain (such as Zinc finger domain) is distinguished.Those skilled in the art can design and not combined with reference to a kind of gene order The zinc finger dna binding structural domain of another kind of highly similar gene order.Select 1.2kb corresponding with collateral line homologue interested Partial gene sequence (Figure 66) subsequently carries out zinc finger dna knot mentioned above as the template for Zinc finger nuclease protein design Close structure domain analysiss.

Embodiment 14：The design of IPP2-K zinc finger dna binding structural domains

It is that IPP2-K zinc fingers select recognition helix using the target site identified for IPP2-K.Zinc finger is shown in table 8 below Design.

Table 8：IPP2-K zinc fingers are designed

ZFN Title

F1

F2

F3

F4

F5

F5

IPP2-K -1072a1

DRSALSR (SEQ ID NO：105)

RNDDRKK (SEQ ID NO：106)

RSDNLST (SEQ ID NO：107)

HSHARIK (SEQ ID NO：108)

RSDVLSE (SEQ ID NO：109)

QSGNLAR (SEQ ID NO：110)

IPP2-K -1072b1

DRSALSR (SEQ ID NO：105)

RNDDRKK (SEQ ID NO：106)

RSDNLAR (SEQ ID NO：111)

TSGSLTR (SEQ ID NO：112)

RSDVLSE (SEQ ID NO：109)

QSGNLAR (SEQ ID NO：110)

IPP2-K -1072c1

DRSALSR (SEQ ID NO：105)

RNDDRKK (SEQ ID NO：106)

TSGNLTR (SEQ ID NO：113)

TSGSLTR (SEQ ID NO：112)

RSDVLSE (SEQ ID NO：109)

QSGNLAR (SEQ ID NO：110)

IPP2-K -r1065a1

RSDHLSE (SEQ ID NO：114)

QSATRKK (SEQ ID NO：115)

ERGTLAR (SEQ ID NO：116)

RSDALTQ (SEQ ID NO：117)

Nothing

Nothing

IPP2-K -r1149a2

RSDSLSA (SEQ ID NO：118)

RSAALAR (SEQ ID NO：119)

RSDNLSE (SEQ ID NO：120)

ASKTRTN (SEQ ID NO：121)

DRSHLAR (SEQ ID NO：122)

Nothing

IPP2-K -1156a2

RSDHLST (SEQ ID NO：123)

QSGSLTR (SEQ ID NO：124)

RSDHLSE (SEQ ID NO：114)

QNHHRIN (SEQ ID NO：125)

TGSNLTR (SEQ ID NO：126)

DRSALAR (SEQ ID NO：127)

The target site that zinc finger is designed is shown in table 9 below.

Table 9：The target site of IPP2-K zinc fingers

ZFN titles	Target site (5 ' to 3 ')
		IPP2-K-1072a1	GAACTGGTTGAGTCGGTC (SEQ ID NO：128)
IPP2-K-1072b1	GAACTGGTTGAGTCGGTC (SEQ ID NO：129)
		IPP2-K-1072c1	GAACTGGTTGAGTCGGTC (SEQ ID NO：129)
IPP2-K-r1065a1	ATGGCCCCACAG (SEQ ID NO：130)
		IPP2-K-r1149a2	GGCACCCAGGTGTTG (SEQ ID NO：131)
IPP2-K-1156a2	GTCGATGGTGGGGTATGG (SEQ ID NO：132)

During IPP2-K to be designed the zinc finger expression vector for mixing protein of the coding with CCHC structures.See above table 1 To table 4.Then by non-standard zinc finger coded sequence and nuclease domain (Wah etc. (1998) of IIS type restriction enzymes FokI Proc.Natl.Acad.Sci.USA 95：The 384-579 amino acids of the sequence of 10564-10569, via 4 aminoacid ZC joints) fusion to be forming IPP2-K ZFN.

Embodiment 15：The intergenic suppression carried out using IPP2-K Zinc finger nucleases

In Urnov (2005) Nature 435 (7042)：646-51 and United States Patent (USP) disclosure No.20050064474 The ability that IPP2-K ZFN described herein promote homologous recombination is tested in (such as embodiment 6-11) described GFP systems.In short, According to the schemes of Invitrogen Lipofectamine 2000, each sample uses 2 μ L Lipofectamine 2000, will The promoterless GFP donors of 50ng every kind of ZFN and 500ng (Urnov (2005) Nature) are transfected into 500,000 report cells In.

The vinblastine of the 0.2 μM of final concentration of addition in 24 hours after transfection, and remove within 72 hours after transfection.

40,000 cells were measured come to cell by each transfection on the desk-top facs analysis instrument of Guava in 5 days after transfection Determine GFP expression.As a result it is shown in Figure 69.

Embodiment 16：C3H1ZFN is expressed in Semen Maydiss HiII cells

A. carrier design

Build the plasmid vector for expressing ZFN albumen in maize cell.Feature zinc finger nucleic acid is formed in order to optimize The expression of 2 different proteins needed for enzyme heterodimer and relative stoichiometric, using strategy is expressed as below, it causes in list The open reading-frame of two kinds of ZFN monomers is inserted on individual carrier, they are driven by single promoter.The strategy is utilized from Thesoa Assigna viruses derivative 2A sequence (Mattion, N.M., Harnish, E.C., Crowley, J.C. and Reilly, P.A. (1996) J.Virol.70,8124-8127), from the Semen Maydiss nuclear localization signal of opaque-2 gene (opaque-2, op-2) (NLS) (Maddaloni, M., DiFonzo, N., Hartings, H., Lazzaroni, N., Salaminil, F., Thompson, And Motto M. (1989) Nucleic Acids Research Vol.17 (18) R.,：7532) and from Semen Maydiss ubiquitin -1 Promoter derived from gene (Christensen A.H., Sharrock R.A., and Quail P.H. (1992) Plant MolBiol.18(4)：Feature 675-89).Progressively module (modular) cloning approach is carried with developing these expression for design Body, for the given paired ZFN encoding genes of any select from library files or de novo synthesis.

First, modify pVAX carriers (to see, for example, United States Patent (USP) disclosure 2005-0267061, include by referring to The disclosure of which) with comprising N-terminal expression structure domain shown in the little figure A-E of Figure 65.Improvement plasmid (pVAX-N2A-NLSop2- EGFP-FokMono) feature of (Figure 65 A) includes redesign and synthesize, coding from NLS derived from Semen Maydiss op-2 (RKRKESNRESARRSRYRK, SEQ IDNO：133) section utilizes Maize codon with redesign and synthesize, coding The section of the FokI nuclease domains of preference.In addition, single nucleotide acid insertion (C) in unique XhoI sites downstreams is created Extra SacI sites are facilitating clone.

Secondly, pVAX carriers (see, for example, United States Patent (USP) disclosure 2005-0267061) are also modified with comprising C-terminal table Up to domain.The feature of the improvement plasmid (pVAX-C2A-NLSop2-EGFP-FokMono) (Figure 65 B) includes redesigning simultaneously Synthesis, coding are from (RKRKESNRESARRSRYRK, the SEQ ID NO of NLS derived from Semen Maydiss op-2：133) section and again The section of the FokI nuclease domains that design and synthesize, coding is had a preference for using Maize codon.In addition, for 2 albumen The subsequent connection of matter encoding domain, in the N-terminal of ZFN ORF the 2A sequences from Thosea asigna viruses are introduced (EGRGSLLTCGDVEENPGP, SEQ ID NO：134).

Termini compatible is produced using restriction enzyme KpnI and BamHI, will encode the ORF's of each zinc finger protein via connection Box gene is cloned in N2A or C2A carriers.Then, will be restricted via identical from the BglII/XhoI fragments of C2A carriers In site insertion N2A carriers, the middle construct comprising following box is produced, the box has NcoI and SacI restricted including flank 2 ZFN encoding domains in site.

Finally, the NcoI/SacI boxes comprising two ZFN genes are built via restrictive diges-tion using those enzymes from the centre Cut out in body (Figure 65 C), and be connected in plasmid backbone pDAB3872 (Figure 65 D).The plasmid of gained includes that ZFN genes add phase Start or stop mover and terminator sequence, add the selection marker for maintaining plasmid.

In final construct (their example is shown in Figure 65 E), ZFN expression cassettes are (including promoter and end Only subcomponent) the flank behaviour that has attL sites and carried out with convenient use Gateway system (Invitrogen, Carlsbad, CA) Make.By the every kind of ZFN constructs generated using the cloning approach be transformed into e.colidh5αcell (Invitrogen, Carlsbad, CA) in, subsequently maintain under suitable selection.

B.DNA is delivered and transient expression

Using the Gigaprep test kits (Qiagen, Valencia, CA) without endonuclease according to manufacturer recommendation Produce in the Bacillus coli cells culture cultivated in the LB culture medium added with antibiotic from 2L and built as described in Figure 65 E The Plasmid Preparation of ZFN expression vectors.Plasmid DNA is directly delivered to Semen Maydiss HiII cultured cells using various methods.

In one example, via Whiskers^TMDNA deliveries are carried out to maize cell.About 24 hours before DNA deliveries, By added with the 3ml PCV HiII Corn suspension cells Secondary Cultures of 7ml conditioned culture medias to 20ml in 125ml In GN6 fluid mediums in Erlenmeyer flasks (lacking the GN6 culture medium of Gelrite), and bottle swingging machine (125rpm, 28 DEG C) on place 24 hours.2mL PCV are taken out, and the GN6S/M added to 12ml in 125ml Erlenmeyer flasks oozes Saturating culture medium (N6 culture medium, 2.0mg/L 2,4-D, 30g/L sucrose, 45.5g/L Sorbitol, 45.5g/L Mannitol, 100mg/L Muscle-inositol, pH6.0).Incubate flask in the case of moderate agitation (125rpm) at 28 DEG C in the dark and reach 30-35 minutes.This During this, made by the way that the GN6S/M fluid mediums of suitable volumes are added into the aseptic whisker (whisker) to weighing in advance Standby 50mg/ml silicon carbide whiskers (Advanced CompositeMaterials, Inc., Eureka Springs, AK) suspends Liquid.After incubating in GN6S/M, the content of each flask is poured in 15mL cone bottom centrifuge tubes.

After cell settlement, about (all but) 1mL GN6S/M liquid is drawn, and collected in 125mL flasks, for not To use.The whisker suspension at full throttle making advance moistening is vortexed 60 seconds, using macropore, filtering type pipet tip general 160 μ L add to centrifuge tube, and add 20 μ g DNA." finger vortex " (" finger vortexed ") is carried out to the pipe, is existed side by side It is placed on Caulk " Vari-Mix II " dental amalgam blender (it is modified to accommodate the culture tube of 17x100mm) In, then stirred 60 seconds with medium speed.After agitation, the mixture of cell, culture medium, whisker and DNA is added together with 18ml GN6 fluid mediums return Erlenmeyer flasks together.Permissive cell on bottle swingging machine (125RPM, 28 DEG C) in the dark Recover 2 hours.

Using the glass cell harvestor unit (glass cell collector unit) being connected with house vacuum pipeline The scattered suspensions of about 5-6mL are filtered to Whatman#4 filter paper (5.5cm) so that each sample obtains 5-6 filter Paper.Filter paper is placed to the GN6 culture medium flat plates of 60x20mm, and is cultivated under dark condition at 28 DEG C.24th, 48 or 72 is little Shi Hou, scrapes the cell from 2-5 filter paper, collects into pipe, is placed on dry ice, then in -80 DEG C of freezings.

In another example that DNA is delivered, the microprojectile bombardment adapted using the scheme from apparatus manufacturer will be pure The Plasmid Preparation without endonuclease changed directly is delivered to maize cell.Use Biolistic PDS-1000/He^TMSystem (Bio-Rad Laboratories, Hercules, CA) carries out all of bombardment.It is for particle coating, 1.0 microns of 3mg is straight The gold grain in footpath 100% ethanol purge 1 time, is cleaned 2 times with sterile distilled water, and the 50 μ l in silication Eppendorf pipes It is resuspended in water.5 μ g plasmid DNA, 20 μ l spermidines (0.1M) and 50 μ l calcium chloride (2.5M) are added to golden suspension.Will mixing Thing is precipitated 10 seconds in incubation at room temperature 10 minutes with 10K rpm, resuspended in 100% 60 μ l cold ethanol, and by 8-9 μ l distribution To each grand carrier (macrocarrier).In order to prepare for bombardment cell, after Secondary Culture 3 days from liquid culture Middle taking-up cell cluster (cell cluster), and trained by the growth of the Mannitol added with each 0.256M and Sorbitol in Pi Shi wares Support it is basis set into osmotic medium on be positioned to the circle of a diameter of 2.5cm.Cell is incubated in osmoticum 4 little before bombardment When.Bombard in instrument mentioned above, this under the vacuum condition of 1100psi and 27 inch of Hg (in of Hg) by inciting somebody to action Organize and be placed on the support of centre and follow workbook to carry out.After treatment the time point of 24 hours, harvests Jing bombardments Cell cluster, freeze in liquid nitrogen, and be stored in -80 DEG C.

DNA is delivered and is involved the utilization of protoplast prepared product with another example of ZFN transient expressions in maize cell.Make With from Mitchell and Petolino (1991) J.Plant.Physiol.137：530-536 and Lyznik etc. (1995) Plant J.8(2)：The method of 177-186 improvement, from HiII corn cell cultures protoplast is prepared.By being centrifuged 5 with 1000rpm Minute, 48 hours (mid log phase) harvests suspension culture after Secondary Culture.Culture medium is removed, and in 10ml W5 trainings Foster base (154mM NaCl₂；125mM CaCl₂·H₂O；5mM KCl₂；5mM glucoses；PH5.8 gentle cleaning 5ml enriches in) PCV。

Cell after 5 minutes are centrifuged with 100rpm to collect cleaning, subsequently incubates in following enzymatic mixture, described Enzymatic mixture filters degerming K3 culture medium (the 2.5g KNO of method in 25ml Jing₃；250mgNH₄NO₃；900mg CaCl₂(two hydrations Thing)；250mg Mg₂SO₄；250mg NH₄SO₄；150mgNaPO₄(single alkali)；250mg xyloses；10ml ferrous sulfate/chelate (chealate) liquid storage (F318)；1ml B5 micronutrients (1000X liquid storage -750mg potassium iodide；250mg molybdic acids (sodium salt) take off Water thing；25mg cobaltous chlorides；25mg copper sulfate)；10ml K3 vitamin (100X liquid storages -1g muscle-inositol；10mg hydrochloric acid pyrroles are trembled Alcohol；100mg thiamine hydrochlorides；10mg nicotinic acid)；+ 0.6M Mannitol；PH5.8 3% cellulase Y-C+0.3% pectin is contained in) Lyase Y23 (pectolyase Y23) (KarlanResearch Products Corp., Cottonwood, AZ).Gently stirring In the case of dynamic (50rpm) 5-6 hours are reached to digest secondary plant cell wall in 25 DEG C of Incubate cells.

After cell wall degradation, the cell strainer (cell strainer) that enzyme-cell mixture flows through 100 microns is filtered, And clean the effluent comprising protoplast and cell debriss with isopyknic K3+0.6M Mannitol culture medium.With 800rpm from Heart protoplast up to 5 minutes, abandoning supernatant, and repeated washing.Cleaning protoplast pellet, and it is sweet in 20ml K3+0.6M It is resuspended in the solution of dew alcohol+9%Ficoll 400.By 10ml, the solution distributes into 2 aseptic plastic pipes, and 2ml TM are cultivated Base (19.52g MES；36.45g Mannitol；40ml 2M CaCl₂·H₂O liquid storages；PH5.5)) leniently it is covered on suspension, Form discontinuous gradient.

It is broken with nonviable protoplast, cell by the protoplast that will be survived for 5 minutes with 800rpm centrifugations Piece and complete suspension cell separate.The different protoplast bands formed in gradient interface are taken out with pipet, and it is new with 10ml Fresh TM solution cleaning, is then centrifuged 5 minutes with 800rpm.The protoplast pellet of resuspended gained in 1mlTM culture medium, and Dyeed come quantitative to the protoplast number that can be survived with 25mg/mg fluorescein(e) diacetates (FDA) in hematimeter. Protoplast solution is adjusted into the 1x10 into TM culture medium⁷The final concentration of individual protoplast/ml.

By about 1x10⁶Individual protoplast (100 μ l) is transferred to the 2ml of the plasmid DNA equipped with 10-80 μ g purification Eppendorf is managed.100 μ l 40%PEG-3350 (Sigma Chemical Co., St.Louis, MO) solution is added dropwise over, and It is gently blended suspension.By protoplast/DNA mixture in incubation at room temperature 30 minutes, then with 1ml GN6 growth mediums Dropwise dilute.Diluted protoplast is incubated 24 hours in the culture medium at 25 DEG C, is subsequently harvested, it is cold in liquid nitrogen Freeze, and be stored in -80 DEG C.

Embodiment 17：Internal ZFN features

In the present embodiment the feature of ZFN is interpreted as including but not limited to ZFN and expresses in the cell of crop species Ability and ZFN by recognize, combine and cut its desired target come in the endogenous gene group for mediating the crop double-strand break The ability split.It will also be appreciated that in the present embodiment, the target of ZFN is in crop gene group in endogenous gene locus and construction Gene.

In order to assess whether through engineering approaches ZFN have the feature for being directed to prediction target gene in genomic context, base is developed In the algoscopy of DNA sequence.The double-strand DNA cleavage induction repair mechanism of prediction ZFN inductions, such as non-homologous end joining (NHEJ) (summary is shown in Cahill etc., (2006) Mechanisms FrontBiosci.1 (11)：1958-76).One of NHEJ As a result it is that the part of DNA breakage chain can be repaired in incomplete mode, causes a small amount of deletion at cleavage site, insertion Or substitute.Those skilled in the art can by various methods to detect DNA sequence in these change.

A. the clone of PCR-based and sequencing

In one example, the Semen Maydiss HiII cultured cells for separating expression ZFN albumen in 24 hours in post-conversion, freezing, and Extracting genome DNA is carried out using Qiagen (Valencia, CA) plant DNeasy extracts kits according to the recommendation of manufacturer. Implement PCR amplifications using the oligonucleotide primers of ZFN cleavage site flanks special to target gene and in prediction.Combination makes With to targeting the special positive PCR primer of IPP2-K gene collateral line homologues (5 '-GGA AGC ATT ATT CCA ATT TGA TGA TAA TGG-3’)(SEQ ID NO：135) with inverse PCR primer (5 '-CCC AAG TGT CGA GGT TGT CAATAT GTT AC-3’)(SEQ ID NO：136) genomic DNA of amplification purification under the following conditions is carried out：25 μ l reaction volumes, in enzyme In the buffer of manufacturer containing 20ng gDNA templates, the every kind of primers of 20pmol, 1%DMSO and 10 unit Accuprime Pf polymerases (Invitrogen, Carlsbad, CA).Amplified production with expected size is derived from by 95 DEG C -1 ', (95 DEG C - 30 ", 61 DEG C -30 ", 72 DEG C -1 ') X 30,72 DEG C -5 ', 4 DEG C-keep composition amplification cycles.

Amplified fragments Direct Cloning is entered into carrier using TA Cloning Kits (Invitrogen, Carlsbad, CA) In pCR2.1 (Invitrogen, Carlsbad, CA).With CEQ Dye Terminator thing cycle sequencing test kit (Beckman Coulter, Fullerton, CA) according to the recommendation of manufacturer separated cloned sequence is sequenced with 96 well formats.In the reality In testing, prediction ZFN 2 short IPP2-K gene specific sequences of protein binding can cut the different of ds-DNA shown in Figure 66 to produce Dimer nuclease.

Analysis to the sequencing result from multiple clones discloses clone #127 comprising just in the ZFN cleavage sites of prediction The little deletion at place, indicates that NHEJ mechanism has mediated the imperfection reparation (Figure 67) of DNA sequence in this site.

These results confirm to induce target at endogenous gene locus of these through engineering approaches ZFN in a specific way in crop species To double-strand break ability.

B. large-scale parallel sequencing analysis

In another example, using the combination of PCR and extensive parallel high temperature sequencing (pyrosequencing) method To inquire the genome of multiple cell samples of the different ZFN albumen of (interrogate) expression targeting this identical sequence.Synthesis Three kinds of variants (5 '-XXX CAC CAA GTT GTATTG CCT TCT CA-3 ') (the SEQ ID NO of positive PCR primer：137) Three kinds of variants (5 '-XX XAT AGG CTT GAG CCA AGC of (wherein XXX=GGG, CCC or GGC) and inverse PCR primer AATCTT-3’)(SEQ ID NO：138) (wherein XXX=GCC, CCG or CGG) (IDT, Coralville, IA).Each primer The 3bp labels at 5 ' ends serve as identifier secret key, and indicate which cell sample amplicon is derived from.It is applied in combination with matching identification The primer pair of symbol label (secret key) is with amplification purification under the following conditions, genomic DNA from maize cell analyte derivative：50 μ l reaction volumes, in the buffer of enzyme manufacturer containing 40ng gDNA templates, the every kind of primers of 20pmol, 1%DMSO and 10 Accuprime Pf polymerases (Invitrogen, Carlsbad, CA) of unit.Amplified production with expected size be derived from by 95 DEG C -1 ', (95 DEG C -30 ", 61 DEG C -30 ", 72 DEG C -1 ') amplification cycles of 30,4 DEG C of X-holding composition, and use MinElute PCR purification kits (Qiagen, Valencia, CA) carry out purification according to the recommendation of manufacturer.

By 454Life Sciences (Branford, CT) such as (Margulies (2005) Nature 437：376- 380) extensive parallel high temperature sequencing reaction (also referred to as 454 sequencing) is directly carried out to PCR primer described in.Included by identification The sequence of the deletion of expected size and location reads out the analysis for implementing 454 sequencing results in DNA molecular.

The result of these analyses indicates there are various little deletions at the expected cleavage site of these ZFN, in such as Figure 68 Shown.These deletions are precisely positioned at ZFN target sites, and indicate that the double-strand induced by ZFN is produced in genome breaks Split, subsequently repaired by NHEJ.These results are further characterized by these through engineering approaches ZFN in a specific way in crop species The ability of the double-strand break of targeting is induced at interior endogenous gene locus.

Embodiment 18：For the Donor DNA Design of targeted integration

In the present embodiment, donor dna is interpreted as including being delivered in plant cell and mix double in Matrix attachment region Ssdna molecule.The mechanism that the incorporation occurs can be via core DNA double center chain broken sites at be independent of the non-same of homology Source end connects (NHEJ；Summary is shown in Cahill etc., (2006) Mechanisms Front Biosci.1：It is 1958-76) or another A kind of similar mechanism.Donor dna drives to the such NHEJ in genome, connection sample is mixed and is referred to as random integration, because for The position of integrating of body DNA is mainly determined by the presence of double-strand DNA cleavage.In the mechanism, donor dna is to the integration in genome The nucleotide sequence of the site that ruptures in genome or the nucleotide sequence of donor itself are not relied on.Therefore, random integration During, " address " of incorporation donor dna is not based on donor DNA sequences regulation or prediction in genome.Random integration It is the main mechanism that donor dna gene transfer occurs during standard plant transformation, the standard plant transformation is via soil bar Bacterium mediation or Biolistic mediation DNA is realized to the delivery in live plant cells.

With random integration conversely, also donor dna can be mixed in genome via targeted integration.Targeted integration is interpreted as Jing By homology dependent mechanism, (summary is shown in van den Bosch etc. for the single-stranded annealing of homology dependency or homologous recombination (2002)Biol Chem.383(6)：873-892) occur in double-strand break site (position).In the fracture of homology dependent DNA In the case of reparation, can mix in this site and include the nucleotide that there is homogeneity or similarity with the DNA at broken site The donor dna of sequence.Therefore, donor dna is integrated into " address " in genome and depends between genome and donor DNA molecule Nucleotide sequence homology or sequence similarity.In botanical system, it is known that the reparation of DNA double center chains fracture using NHEJ and (summary is shown in Puchta (2005) J.Exp.Bot.56 to both homology dependent pathways：1-14).

In the present embodiment, we describe via in sequence-specific ZFN protein induced double-strand break site Targeted integration and the design of donor DNA molecule that to be integrated in genome and structure.Different ZFN albumen can be in target gene sequence Double-strand break is induced at different nucleotide in row；The specific site of the double-strand break for being induced is referred to as position.

As described in example 13 above, we have characterized the nucleotide sequence of the target gene IPP2K from Semen Maydiss.With Afterwards, we devise the ZFN albumen (embodiment 14) with reference to the target gene particular bases, and confirm it in two kinds of Heterologous Systems Target gene in the sequence at and for the combination/cleavage activity (embodiment 15-17) of endogenous gene in maize cell.This In, we describe the double-strand break position incorporation Semen Maydiss for being designed for the ZFN mediations in IPP2K genes via targeted integration The structure of the various donor molecules in genome.For known to nucleotide sequence and predicting that the sequence is any comprising double-strand break Any position in genome, those skilled in the art can build and be designed for being incorporated via the targeted integration that homology drives Enter the donor DNA molecule in the double-strand break of ZFN inductions.

In an embodiment as herein described, donor DNA molecule is included with the target gene IPP2K's for targeting position Nucleotide sequence identical nucleotide sequence section is the autonomous herbicide tolerance genes expression cassette on boundary.In this embodiment, Autonomous herbicide tolerant box is interpreted as including complete promoter-transcript unit (PTU) that it includes promoter, herbicide tolerant base Cause and it is known be functional terminator sequence in plant cell.Those skilled in the art may be selected any promoter, gene Autonomous PTU is constituted with sub-portfolio is terminated.Also included on the Plasmid Constructs is target gene with position shown in Semen Maydiss (IPP2K) DNA fragmentation with sequence iden.These fragments serve as " the homology flank " of donor dna, and whole via targeting Conjunction is directly incorporated into the donor in target gene in specified location.PTU upstream and downstream with relative to correct 5 ' -3 ' of PTU Orientation places homology flank.Skilled in the art realises that donor dna has the homology of different size and orientation in building Flank.

In another embodiment as herein described, donor DNA molecule include following plasmid construction, its include with target The IPP2K nucleotide sequence identical nucleotide sequences section of position is the non-autonomous herbicide tolerance genes expression cassette on boundary. In the embodiment, non-autonomous herbicide tolerant box is interpreted as the imperfect promoter-transcription for including lacking functional promoter Unit (PTU).Non-autonomous PTU really comprising herbicide tolerance genes and it is known be functional terminator sequence in plant cell Row.Those skilled in the art may be selected any gene and termination sub-portfolio to constitute non-autonomous PTU.In the example of non-autonomous donor In son, the expression of herbicide tolerance genes depends on donor section to mixing in the genomic locations being close to functional promoter Enter, the functional promoter can drive the expression of the gene.Can be appreciated that relatively rare following situation, wherein for know from experience via Random integration and mixing wherein exist promoter serendipitous and the promoter can be used to drive herbicide tolerance genes expression Genetic loci in.Or, the interior target gene with ad-hoc location in Semen Maydiss is built based on donor dna there is sequence iden , the presence of the homology flank of the DNA fragmentation of appropriate length, donor dna can occur to the essence in the target gene of ad-hoc location True targeted integration (as described in regard to autonomous donor), therefore using the endogenesis promoter of the target gene.In the embodiment In, place homology flank to be orientated relative to correct 5 ' -3 ' of PTU in the upstream and downstream of PTU.Those skilled in the art connect Connect the homology flank during donor dna builds with different size and orientation.

In two embodiments as herein described (design of autonomous and non-autonomous donor), plasmid construction is typically wrapped Containing realize herbicide tolerance genes clone, expression and with other element of post analysis.This class component include bacterial origin of replication, Restriction site of transformation etc., and it is on the books below.Skilled in the art realises that constituting the difference unit of donor DNA molecule The utilization of part.

A. bacterial isolateses and condition of culture

Using Luria-Bertani meat soup (LB：10g/L antibacterial culturing tryptones, 10g/L NaCl, 5g/L antibacterials Culture yeast extract), LB agar (added with the LB meat soups of 15g/L Bacto-agars) or Terrific meat soup (TB： 12g/L antibacterial culturing tryptones, 24g/L Bacto-yeast extracts, 0.4%v/v glycerol, 17mM KH₂PO₄、 72mM K₂HPO₄) cultivate coli strain (One at 37 DEG CThe Competent cells of Top 10；MAX DH5α^TMCompetent cell, Invitrogen Life Technologies, Carlsbad, CA) reach 16 hours.Liquid culture is shaken with 200rpm.When needed by chloromycetin (50 μ g/ml), kanamycin (50 μ g/ml) or Ampicillin (100 μ g/ml) adds to culture medium.All antibiotic, culture medium and buffer reagent used in this research are equal Purchased from Sigma-Aldrich Corporation (St.Louis, MO) or Difco Laboratories (Detroit, MI).

B. plasmid backbone position -1

By comprising IPP2K positions -1 homology flank plasmid backbone be transformed into allow any donor DNA sequences are whole In being bonded to the respective target site of IPP2K genes.Skilled in the art realises that using various cloning sites, module design element and The plasmid backbone of the sequence homologous with any target sequence in gene of interest group.Here the plasmid backbone for illustrating is by Basic plasmid Carrier pBC SK (-) phasmids (3.4Kbp) (Stratagene, La Jolla, CA) starts.Come using the synthesis of hereinafter described 4 steps Build the plasmid backbone of position -1.

In step #1, Basic plasmid is prepared.In 37 DEG C of uses, 10 unit Spe I and 10 unit Not I (New England Biolabs, Beverly, MA) restriction endonuclease made 3 μ g pBCSK (-) linearisations up to 1 hour.Mending 1.0%TAE (0.04M filled with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St.Louis, MO) Tris- acetates, 0.002M EDTA) in agarose gel in 100V to restricted DNA electrophoresis 1 hour.Manifested with ultraviolet DNA fragmentation, and by comparing to estimate with 1Kbp DNA ladders (Invitrogen Life Technologies, Carlsbad, CA) Meter clip size.Gel cutting is carried out to subcloning vector pBC SK (-) of 3.4Kbp Jing Spe I/Not I digestion, and is used QIA quick gel extraction kits (QIAquick Gel Extraction Kit, QIAGEN Inc., Valencia, CA) according to Guidance according to manufacturer carries out purification.

In step #2,5 ' and 3 ' the homology flanks from IPP2K positions -1 are separated.By IntegratedDNA Technologies, Inc. (Coralville, IA) synthesize following oligonucleotide primer under the conditions of standard desalination, and dilute with water Release to the concentration of 0.125 μ g/ul：

5’-GCGGCCGCGTCTCACCGCGGCTTGGGGATTGGATACGGAGCT-3’(SEQ ID NO：143)

5’-ACTAGTGATATGGCCCCACAGGAGTTGCTCATGACTTG-3’(SEQ IDNO：144)

5’-ACTAGTCCAGAACTGGTTGAGTCGGTCAAACAAGATTGCT-3’(SEQID NO：145)

5’-GTCGACCTTGATGCTACCCATTGGGCTGTTGT-3’(SEQ ID NO：146).

Using by TaKaRa Biotechnology Inc., Seta 3-4-1, Otsu, Shiga, 520-2193, Japan The reagent of offer implements pcr amplification reaction, and its composition is as follows：5μl 10X LA PCR^TMBuffer II (Mg²⁺), 20ng double-strands GDNA templates (Semen Maydiss HiII), 10pmol forward direction oligonucleotide primers, 10pmol reverse oligonucleotide primers, 8 μ l dNTP mixing Thing (each 2.5mM), 33.5 μ l H₂O, 0.5 μ l (2.5 units) TaKaRa LA Taq^TMArchaeal dna polymerase, 1 drop mineral oil.Make Enter performing PCR reaction under following cycling condition with Perkin-Elmer Cetus48 sample DNA thermal cyclers (Norwalk, CT)： 94 DEG C of 4 minutes/1 circulations；98 DEG C 20 seconds, 65 DEG C 1 minute, 68 DEG C 1 minute/30 circulation；72 DEG C, 5 minutes/1 circulation；4 DEG C/keep.In 100V to each PCR reaction electricity of 15 μ l in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Swimming 1 hour.Manifest amplified fragments with ultraviolet, and estimate clip size by comparing with 1KbpDNA ladders.According to 0.821Kbp The presence of (5 ' homology flank) or 0.821Kbp (3 ' homology flank) DNA fragmentation is judging expected amplified production.

These fragments are carried out with gel cutting, and using QIA quick gel extraction kits (QIAGENInc., Valencia, CA) carry out purification according to the guidance of manufacturer.Then TOPO TA are usedTest kit (contains2.1 carriers) and OneTOP10 Competent Bacillus coli cells (Invitrogen Life Technologies, Carlsbad, CA) fragment of purification is cloned in pCR2.1 plasmids according to the scheme of manufacturer.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml kanamycin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Spe I and Not I are digested from the detached 3 μ g plasmids of 5 ' homology flank cloned plasmids.With 10 unit Spe I and 20 unit Sal I (NewEngland Biolabs, Beverly, MA) digest 3 ' homology flank cloned plasmids.Will be all of Plasmid digestions are incubated 1 hour at 37 DEG C.At 100V pair in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Restricted DNA electrophoresis 1 hour.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to 3.9KbpThe insertion of 0.821Kbp (5 ' homology flank) or 0.821Kbp (3 ' homology flank) outside 2.1 carriers The presence of DNA fragmentation is judging expected plasmid cloning.

Using CEQ^TMDTCS- quickly starts test kit (CEQ^TMDTCS-Quick Start Kit, Beckman- Coulter, Palo Alto, CA) the double-strand sequencing reaction of enforcement plasmid cloning as described in manufacturer.Use Performa DTR gel filtration cylinders (Performa DTR Gel FiltrationCartridges, Edge BioSystems, Gaithersburg, MD) purification reaction as described in the scheme of manufacturer.In Beckman-Coulter CEQ^TM Analytical sequence reaction in 2000XL DNA analysis systems, and use Sequencher^TM4.1.4 version (Gene Codes Corporation, Ann Arbor, MI) carrying out nucleotide sign.Show in Figure 87 with from position -1 derived from IPP2K Sequence (the SEQ ID NO of the corresponding 0.821Kbp fragments of 5 ' homology flanks：171).Show in Figure 88 and spread out from IPP2K Sequence (the SEQ ID NO of the raw corresponding 0.821Kbp fragments of the homology flank of position -1 3 '：172).

In step #3, the homology flank of position -1 5 ' is connected in Basic plasmid.Pair with include tram -1 The corresponding restriction fragment of clone of 5 ' homology flanking sequences carries out gel cutting, and using QIA PhastGel extracts reagents Box (QIAGEN Inc., Valencia, CA) carries out purification according to the guidance of manufacturer.Then connected using 500 units T4DNA Connecing enzyme (Invitrogen Life Technologies, Carlsbad, CA) will be with corresponding of -1 5 ' homology flank of position Section (0.821Kbp) is with 1: 5 carrier: insert ratio incubation condition of 16 hours in 16 DEG C of water-baths in 20 μ l reaction volumes Under be connected to SpeI/Not I digest purification Basic plasmid (step #1).Subsequently 5 μ l coupled reactions are converted to large intestine Bacillus OneThe Competent cells of Top 10 (Invitrogen Life Technologies, Carlsbad, CA) In, and the bed board under the alternative condition described in manufacturer.By individual colonies be seeded to 14ml containing 2ml be supplemented with 50 μ l/ml cards that In Falcon pipes (Becton-Dickinson, Franklin Lakes, NJ) of the TB of mycin, and in the feelings shaken with 200rpm Incubate 16 hours at 37 DEG C under condition.

After incubation, by 1.5ml cells be transferred to 1.7ml Costar microcentrifugal tubes (Fisher Scientific, Pittsburgh, PA) in, and precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid reagent Box (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) separation quality grain as above DNA.The detached plasmids of 3 μ g are digested with 10 unit Spe I and Not I (New EnglandBiolabs, Beverly, MA) DNA, and incubate 1 hour at 37 DEG C.In 100V to limiting in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Property DNA electrophoresis 1 hour.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to The presence of 0.821Kbp insertions DNA fragmentation (5 ' homology flank) outside 3.4Kbp Basic plasmids is judging expected plasmid gram It is grand.

In step #4, the homology flank of position -1 3 ' is connected in the product of step #3.In 37 DEG C of uses, 10 lists Position Spe I and 20 unit Sal I (New England Biolabs, Beverly, MA) restriction endonucleases were up to 1 hour To make to transform product linearisation described in 3 μ g steps #3.It is being supplemented with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St.Louis, MO) 1.0%TAE (0.04M Tris- acetates, 0.002M EDTA) agarose gel in In 100V to restricted DNA electrophoresis 1 hour.Manifest DNA fragmentation with ultraviolet, and by with 1Kbp DNA ladder (Invitrogen Life Technologies, Carlsbad, CA) compare to estimate clip size.About 4.2Kbp Jing Spe I/Sal I are digested The product from step #3 carry out gel cutting, and using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) carry out purification according to the guidance of manufacturer.

Subsequently use 1: 5 carrier: insert ratio and 500 unit T4DNA ligase (InvitrogenLife Technologies, Carlsbad, CA) by step #2 generate 3 ' homology flank donors isolated fragment (0.821Kbp) Mix in 20 μ l coupled reactions with #3 products the step of digesting simultaneously purification with Spe I/Sal I as described above.To connect anti- Should incubate 16 hours in 16 DEG C of water-baths.After connection, 5 μ l coupled reactions are converted to MAX DH5α^TMChange In learning competent cell (Invitrogen LifeTechnologies, Carlsbad, CA), this enters according to the recommendation of manufacturer OK.Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml chloromycetin containing 2ml Dickinson, Franklin Lakes, NJ) in.

Culture is incubated 16 hours in the case of 200rpm shakes at 37 DEG C.After incubation, 1.5ml cells are transferred to In 1.7ml Costar microcentrifugal tubes (Fisher Scientific, Pittsburgh, PA), and 1 point is precipitated with 16,000xg Clock.Supernatant is removed, and is usedPlasmid kit (BDBiosciences/Clontech/Macherey- Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Sal I and Not I (New England Biolabs, Beverly, MA) the detached plasmids of 3 μ g of digestion, and incubate 1 hour at 37 DEG C.It is being supplemented with 0.5% In 100V to restricted DNA electrophoresis 1 hour in the 1.0%TAE agarose gel of ethidium bromide.Manifest fragment with ultraviolet, and Estimate clip size by comparing with 1Kbp DNA ladders.According to the two of 1.64Kbp (insert) and 3.33Kbp (Basic plasmid) The presence of kind of DNA fragmentation is judging to be expected clone.The plasmid of gained is named as pDAB7471 (Figure 70).

C. plasmid backbone position -2

By comprising IPP2K positions -2 homology flank plasmid backbone be transformed into allow any donor DNA sequences are whole In being bonded to the respective target site of IPP2K genes.Skilled in the art realises that using various cloning sites, module design element and The plasmid backbone of the sequence homologous with any target sequence in gene of interest group.Here the plasmid backbone for illustrating is by Basic plasmid Carrier pBC SK (-) phasmids (3.4Kbp) (Stratagene, La Jolla, CA) starts.Come using the synthesis of hereinafter described 4 steps Build the plasmid backbone of position -2.

In step #1, Basic plasmid is prepared.In 37 DEG C of uses, 10 unit Spe I and 10 unit Not I (New England Biolabs, Beverly, MA) restriction endonuclease made 3 μ g pBCSK (-) linearisations up to 1 hour.Mending 1.0%TAE (0.04M filled with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St.Louis, MO) Tris- acetates, 0.002M EDTA) in agarose gel in 100V to restricted DNA electrophoresis 1 hour.Manifested with ultraviolet DNA fragmentation, and by comparing to estimate with 1Kbp DNA ladders (Invitrogen Life Technologies, Carlsbad, CA) Meter clip size.Gel cutting is carried out to subcloning vector pBC SK (-) of 3.4Kbp Jing Spe I/Not I digestion, and is used QIA quick gel extraction kits (QIAquick Gel Extraction Kit, QIAGEN Inc., Valencia, CA) according to Guidance according to manufacturer carries out purification.

In step #2,5 ' and 3 ' the homology flanks from IPP2K positions -2 are separated.By IntegratedDNA Technologies, Inc. (Coralville, IA) synthesize following oligonucleotide primer under the conditions of standard desalination, and dilute with water Release to the concentration of 0.125 μ g/ul：

5’-GCGGCCGCTAGATAGCAGATGCAGATTGCT-3’(SEQ ID NO：147)

5’-ACTAGTATTGGCACCCAGGTGTTGGCTCA-3’(SEQ ID NO：148)

5’-ACTAGTCATGTCGATGGTGGGGTATGGTTCAGATTCAG-3’(SEQ IDNO：149)

5’-GTCGACGTACAATGATTTCAGGTTACGGCCTCAGGAC-3’(SEQ IDNO：150).

Using by TaKaRa Biotechnology Inc., Seta 3-4-1, Otsu, Shiga, 520-2193, Japan The reagent of offer implements pcr amplification reaction, and its composition is as follows：5μl 10X LA PCR^TMBuffer II (Mg²⁺), 20ng double-strands GDNA templates (Semen Maydiss HiII), 10pmol forward direction oligonucleotide primers, 10pmol reverse oligonucleotide primers, 8 μ l dNTP mixing Thing (each 2.5mM), 33.5 μ l H₂O, 0.5 μ l (2.5 units) TaKaRa LA Taq^TMArchaeal dna polymerase, 1 drop mineral oil.Make Implement PCR reactions under following cycling condition with Perkin-Elmer Cetus48 sample DNA thermal cyclers (Norwalk, CT)： 94 DEG C of 4 minutes/1 circulations；98 DEG C 20 seconds, 55 DEG C 1 minute, 68 DEG C 1 minute/30 circulation；72 DEG C of 5 minutes/1 circulations；4 DEG C/keep.In 100V to each PCR reaction electricity of 15 μ l in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Swimming 1 hour.Manifest amplified fragments with ultraviolet, and estimate clip size by comparing with 1KbpDNA ladders.According to 0.855Kbp The presence of (5 ' homology flank) or 0.845Kbp (3 ' homology flank) DNA fragmentation is judging expected amplified production.To this A little fragments carry out gel cutting, and using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) according to system Making the guidance of business carries out purification.Then TOPO TA are usedTest kit (contains2.1 carriers) and OneThe Competent Bacillus coli cells of TOP 10 (Invitrogen Life Technologies, Carlsbad, CA) The fragment of purification is cloned in pCR2.1 plasmids according to the scheme of manufacturer.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml kanamycin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Spe I and Not I are digested from the detached 3 μ g plasmids of 5 ' homology flank cloned plasmids.With 10 unit Spe I and 20 unit Sal I (NewEngland Biolabs, Beverly, MA) digest 3 ' homology flank cloned plasmids (threeprime-homology flank clone plasmid).All of plasmid digestions are incubated 1 hour at 37 DEG C.

It is little to restricted DNA electrophoresis 1 in 100V in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide When.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to 3.9Kbp The presence of the insertion DNA fragmentation of the 0.855Kbp (5 ' homology flank) or 0.845Kbp (3 ' homology flank) outside 2.1 carriers To judge expected plasmid cloning.

Using CEQ^TMDTCS- quickly starts test kit (CEQ^TMDTCS-Quick Start Kit, Beckman- Coulter, Palo Alto, CA) the double-strand sequencing reaction of enforcement plasmid cloning as described in manufacturer.Use Performa DTR gel filtration cylinders (Performa DTR Gel FiltrationCartridges, Edge BioSystems, Gaithersburg, MD) purification reaction as described in the scheme of manufacturer.In Beckman-Coulter CEQ^TM Analytical sequence reaction in 2000XL DNA analysis systems, and use Sequencher^TM4.1.4 version (Gene Codes Corporation, Ann Arbor, MI) carrying out nucleotide sign.Show in Figure 89 with from position -2 derived from IPP2K Sequence (the SEQ ID NO of the corresponding 0.855Kbp fragments of 5 ' homology flanks：139).Show in Figure 90 and spread out from IPP2K Sequence (the SEQ ID NO of the raw corresponding 0.845Kbp fragments of the homology flank of position -2 3 '：140).

In step #3, the homology flank of position -1 5 ' is connected in Basic plasmid.Pair with include tram -2 The corresponding restriction fragment of clone of 5 ' homology flanking sequences carries out gel cutting, and using QIA PhastGel extracts reagents Box (QIAGEN Inc., Valencia, CA) carries out purification according to the guidance of manufacturer.Then connected using 500 units T4DNA Connecing enzyme (Invitrogen Life Technologies, Carlsbad, CA) will be with corresponding of -1 5 ' homology flank of position Section (0.855Kbp) is with 1: 5 carrier: insert ratio incubation condition of 16 hours in 16 DEG C of water-baths in 20 μ l reaction volumes Under be connected to SpeI/Not I digest purification Basic plasmid (step #1).

Subsequently 5 μ l coupled reactions are converted to escherichia coli OneThe Competent cells of Top 10 In (Invitrogen Life Technologies, Carlsbad, CA), and the bed board under the alternative condition described in manufacturer. Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml kanamycin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Spe I and Not I (New EnglandBiolabs, Beverly, MA) digest the detached plasmid DNA of 3 μ g, and 37 DEG C incubate 1 hour.It is electric to restricted DNA in 100V in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Swimming 1 hour.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.It is basic according to 3.4Kbp The presence of 0.855Kbp insertions DNA fragmentation (5 ' homology flank) outside plasmid is judging expected plasmid cloning.

In step #4, the homology flank of position -2 3 ' is connected in the product of step #3.In 37 DEG C of uses, 10 lists Position Spe I and 20 unit Sal I (New England Biolabs, Beverly, MA) restriction endonucleases were up to 1 hour To make to transform product linearisation described in 3 μ g steps #3.It is being supplemented with 0.5% ethidium bromide (Sigma-Aldrich Corporation, St.Louis, MO) 1.0%TAE (0.04M Tris- acetates, 0.002M EDTA) agarose gel in In 100V to restricted DNA electrophoresis 1 hour.Manifest DNA fragmentation with ultraviolet, and by with 1Kbp DNA ladder (Invitrogen Life Technologies, Carlsbad, CA) compare to estimate clip size.To 4.25Kbp Jing Spe I/Sal I digestion Carry out gel cutting from the product of step #3, and using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) carry out purification according to the guidance of manufacturer.

Subsequently use 1: 5 carrier: insert ratio and 500 unit T4DNA ligase (InvitrogenLife Technologies, Carlsbad, CA) by step #2 generate 3 ' homology flank donors isolated fragment (0.845Kbp) Mix in 20 μ l coupled reactions with #3 products the step of digesting simultaneously purification with Spe I/Sal I as described above.To connect anti- Should incubate 16 hours in 16 DEG C of water-baths.After connection, 5 μ l coupled reactions are converted to MAX DH5α^TMChange In learning competent cell (Invitrogen LifeTechnologies, Carlsbad, CA), this enters according to the recommendation of manufacturer OK.Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml chloromycetin containing 2ml Dickinson, Franklin Lakes, NJ) in.Culture is incubated 16 hours in the case of 200rpm shakes at 37 DEG C. After incubation, by 1.5ml cells be transferred to 1.7ml Costar microcentrifugal tubes (Fisher Scientific, Pittsburgh, PA in), and precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Sal I and Not I (New EnglandBiolabs, Beverly, MA) digest the detached plasmids of 3 μ g, and at 37 DEG C Incubate 1 hour.It is little to restricted DNA electrophoresis 1 in 100V in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide When.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to about 1.7Kbp (insert) Presence with two kinds of DNA fragmentations of 3.33Kbp (Basic plasmid) is judging to be expected clone.The plasmid of gained is named as pDAB7451 (Figure 71).

D. autonomous herbicide tolerance genes expression cassette builds

The autonomous herbicide tolerance genes expression cassette (Figure 72) comprising complete promoter-transcript unit (PTU) is built, it is described Complete promoter-transcript unit (PTU) terminates comprising promoter, herbicide tolerance genes and polyadenylation (polyadenylic acid) Sequence.In this embodiment, promoter sequence is derived from the [(Plant such as McElroy of rice (O.sativa) actin 1 Cell 2,163-171；1990)；GenBank accession number S44221 and GenBank accession number X63830].Herbicide tolerance genes Comprising PAT (phosphinothricin acetyl transferase) gene, it is (initially chromogenic from green that it gives the resistance to herbicide Bialaphos PAT coding regions (GenBank accession number M22827 derived from streptomycete (Streptomyces viridochromogenes)； The Gene such as Wohlleben 70,25-37；1988) modification pattern).To repairing for the initial sequence of the most long open reading-frame of M22827 Decorations are substantial, and including the expression changed during codon utilizes pattern to optimize plant.Except with methionine replace figured silk fabrics ammonia Acid as first coded amino acid, and outside adding alanine as second coded amino acid, can from the PAT of pDAB3014 The protein of frame coding is identical with the protein that the most long open reading-frame of accession number M22827 is encoded.With GenBank accession number What I43995 found PAT builds again (rebulit) pattern.Terminator sequence is derived from Semen Maydiss (Z.mays L.) lipase [the maize lipase cDNA clone of GenBank accession number L35913 simply replaces L35913 with the 2468th G in pDAB3014 1093rd C.The Semen Maydiss sequence is that pat gene includes 3 ' untranslated regions/tanscription termination sub-district].

By Integrated DNA Technologies, Inc. (Coralville, IA) synthesizes under the conditions of standard desalination Following oligonucleotide primer, and it is diluted with water to 0.125 μ g/ μ l concentration：

5’-ACTAGTTAACTGACCTCACTCGAGGTCATTCATATGCTTGA-3’(SEQID NO：151)

5’-ACTAGTGTGAATTCAGCACTTAAAGATCT-3’(SEQ ID NO：152).

Using by TaKaRa Biotechnology Inc., Seta 3-4-1, Otsu, Shiga, 520-2193, Japan The reagent of offer implements pcr amplification reaction, and its composition is as follows：5μl 10X LA PCR^TMBuffer II (Mg²⁺), 20ng double-strand moulds Plate [pDAB3014 plasmid DNA], 10pmol forward direction oligonucleotide primers, 10pmol reverse oligonucleotide primers, 8 μ l dNTP are mixed Compound (each 2.5mM), 33.5 μ l H₂O, 0.5 μ l (2.5 units) TaKaRa LA Taq^TMArchaeal dna polymerase, 1 drop mineral oil. Implement PCR under following cycling condition using the sample DNA thermal cyclers (Norwalk, CT) of Perkin-ElmerCetus 48 anti- Should：94 DEG C of 4 minutes/1 circulations；98 DEG C 20 seconds, 55 DEG C 1 minute, 68 DEG C 3 minutes/30 circulation；72 DEG C of 5 minutes/1 circulations； 4 DEG C/keep.In 100V to each PCR reaction electricity of 15 μ l in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Swimming 1 hour.

Manifest amplified fragments with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to 2.3Kbp The presence of DNA fragmentation is judging expected amplified production.Gel cutting is carried out to the fragment, and is extracted using QIA PhastGels Test kit (QIAGEN Inc., Valencia, CA) carries out purification according to the guidance of manufacturer.Then TOPO TA are usedTest kit is cloned into purified fragments in pCR2.1 plasmids, and converts to OneTOP10 chemoreceptions In state Bacillus coli cells (Invitrogen Life Technologies, Carlsbad, CA), according to the scheme of manufacturer Carry out.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml kanamycin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Spe I and Not I digest the detached plasmids of 3 μ g.All plasmid digestions are incubated 1 hour at 37 DEG C.

It is little to restricted DNA electrophoresis 1 in 100V in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide When.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to 3.9Kbp The presence of the insertion DNA fragmentation of the 2.325Kbp outside 2.1 carriers is judging expected plasmid cloning.Using CEQ^TMDTCS- is quick Start test kit (Beckman-Coulter, Palo Alto, CA) implements plasmid cloning double-strand as described in manufacturer Sequencing reaction.Using Performa DTR gel filtration cylinders (Edge BioSystems, Gaithersburg, MD) such as manufacturer Scheme described in as purification reaction.In Beckman-Coulter CEQ^TMAnalytical sequence in 2000XL DNA analysis systems Reaction, and use Sequencher^TM4.1.4 version (Gene Codes Corporation, Ann Arbor, MI) is carrying out nucleoside Acid is characterized.

E. autonomous herbicide tolerance genes box inserts plasmid backbone --- in autonomous donor

In order to create donor plasmid, by insertion embodiment 18B of autonomous herbicide tolerance genes box described in embodiment 18D and In plasmid backbone construct described in 18C.To the restricted of the clonal derivation of self-contained expected 2.325Kbp sequences mentioned above Fragment (Figure 72) carries out gel cutting, and using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) according to Purification is carried out according to the guidance of manufacturer.

Then by the fragment and the pDAB7471 (positions for having used restriction enzyme Spe I digestion and subsequent dephosphorylized purification Put -1 plasmid backbone, Figure 70) or pDAB7451 (plasmid backbone of position -2, Figure 71) combine in coupled reaction.In following condition It is lower to implement connection：1: 5 carrier in the reaction volume of 20 μ l: insert ratio and 500 unit T4DNA ligases (Invitrogen Life Technologies, Carlsbad, CA), under conditions of incubating 16 hours in 16 DEG C of water-baths.With 5 μ l coupled reactions are converted to 50 μ l escherichia coli MAX afterwards DH5α^TMCompetent cell In (Invitrogen LifeTechnologies, Carlsbad, CA), and the bed board under alternative condition described in manufacturer.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml chloromycetin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Spe I (New England Biolabs, Beverly, MA) digest the detached plasmid DNA of 3 μ g, and in 37 DEG C of temperature Educate 1 hour.It is little to restricted DNA electrophoresis 1 in 100V in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide When.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to 2.325Kbp peace treaties The presence of 4.9Kbp (pDAB7471 carriers) or 2.325Kbp and about 5.0Kbp (pDAB7451 carriers) DNA fragmentation come judge be expected Plasmid cloning.

The plasmid of gained is respectively designated as pDAB7422 (the autonomous donor in position -1), and (Figure 73) (position -2 is certainly with pDAB7452 Main donor) (Figure 74).

F. non-autonomous herbicide tolerance genes expression cassette builds

Build the non-autonomous herbicide tolerance genes expression cassette (Figure 75) comprising imperfect promoter-transcript unit (PTU). In this embodiment, using using from the derivative 2A sequences of Thesoa assigna viruses (Mattion, N.M., Harnish, E.C., Crowley, J.C. and Reilly, P.A. (1996) J.Virol.70,8124-8127), herbicide tolerance genes and poly- Functional strategy of polyadenylation (polyadenylic acid) terminator sequence (but without promoter).In this embodiment, 2A translations Termination signal sequence have been modified to herbicide tolerance genes to meet reading frame in the way of translate.In addition, 2A/ herbicides Coded sequence has been modified to be consistent with the translation reading frame of IPP2K gene targets.Herbicide tolerance genes include PAT (phosphines Silk rhzomorph Acetylase) gene, it is (initially derivative from green color-producing streptomycete that it gives the resistance to herbicide Bialaphos PAT coding regions (GenBank accession number M22827；Wohlleben etc., Gene 70,25-37；1988) modification pattern).It is right The modification of the initial sequence of the most long open reading-frame of M22827 is substantial, and utilizes pattern to optimize plant including codon is changed Expression in thing.Except L-Valine is replaced as first coded amino acid with methionine, and add alanine as second Outside aminoacid, (it starts from the most long open reading-frame of the protein encoded from the PAT open reading-frames of pDAB3014 and M22827 The GTG that M22827 is the 244th) coding protein it is identical.Building again for PAT is found with GenBank accession number I43995 (rebulit) pattern.Terminator sequence is derived from the Semen Maydiss lipase [maize lipase of GenBank accession number L35913 CDNA clone, simply replaces the 1093rd C of L35913 with the 2468th G in pDAB3014].The Semen Maydiss sequence is pat gene bag Containing 3 ' untranslated regions/tanscription termination sub-district.

By Integrated DNA Technologies, Inc. (Coralville, IA) synthesizes under the conditions of standard desalination Following oligonucleotide primer, and it is diluted with water to 0.125 μ g/ μ l concentration：

5’-ACTAGTGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGC CCTAGGATGGCTTCTCCGGAGAGGAGACCAGTTGA-3(SEQ ID NO：153)

5’-ACTAGTATGCATGTGAATTCAGCACTTAAAGATCT-3’(SEQ IDNO：154).

Using by TaKaRa Biotechnology Inc. (Seta 3-4-1, Otsu, Shiga, 520-2193, Japan) The reagent of offer implements pcr amplification reaction, and its composition is as follows：5μl 10X LA PCR^TMBuffer II (Mg²⁺), 20ng double-strand moulds Plate (pDAB3014 plasmid DNA), 10pmol forward direction oligonucleotide primers, 10pmol reverse oligonucleotide primers, 8 μ l dNTP are mixed Compound (each 2.5mM), 33.5 μ l H₂O, 0.5 μ l (2.5 units) TaKaRa LA Taq^TMArchaeal dna polymerase, 1 drop mineral oil. Implement PCR under following cycling condition using the sample DNA thermal cyclers (Norwalk, CT) of Perkin-ElmerCetus 48 anti- Should：94 DEG C of 4 minutes/1 circulations；98 DEG C 20 seconds, 55 DEG C 1 minute, 68 DEG C 2 minutes/30 circulation；72 DEG C of 5 minutes/1 circulations； 4 DEG C/keep.In 100V to each PCR reaction electricity of 15 μ l in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Swimming 1 hour.Manifest amplified fragments with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to about 1Kbp The presence of DNA fragmentation is judging expected amplified production.Gel cutting is carried out to the fragment, and is extracted using QIA PhastGels Test kit (QIAGEN Inc., Valencia, CA) carries out purification according to the guidance of manufacturer.Then TOPO TA are usedTest kit is cloned into purified fragments in pCR2.1 plasmids, and converts to OneThe chemoreceptions of TOP 10 In state Bacillus coli cells (Invitrogen Life Technologies, Carlsbad, CA), according to the scheme of manufacturer Carry out.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml kanamycin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000x g.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 unit Spe I digest the detached plasmids of 3 μ g.All plasmid digestions are incubated 1 hour at 37 DEG C.It is being supplemented with 0.5% In 100V to restricted DNA electrophoresis 1 hour in the 1.0%TAE agarose gel of ethidium bromide.Manifest fragment with ultraviolet, and Estimate clip size by comparing with 1Kbp DNA ladders.According to about 1.0Kbp insert DNA fragmentation and 3.9Kbp ( 2.1 Carrier) presence judging expected plasmid cloning.Using CEQ^TMDTCS- quickly start test kit (Beckman-Coulter, Palo Alto, CA) the double-strand sequencing reaction of enforcement plasmid cloning as described in manufacturer.It is solidifying using Performa DTR Glue cartridge filter (Edge BioSystems, Gaithersburg, MD) purification reaction as described in the scheme of manufacturer. Beckman-CoulterCEQ^TMAnalytical sequence reaction in 2000XL DNA analysis systems, and use Sequencher^TM 4.1.4 Version (Gene Codes Corporation, Ann Arbor, MI) is carrying out nucleotide sign.

G. non-autonomous herbicide tolerance genes box inserts plasmid backbone --- in non-autonomous donor

In order to create donor plasmid, non-autonomous herbicide tolerance genes box described in embodiment 18F is inserted into embodiment 18B In plasmid backbone construct described in 18C.Pair carry out with the corresponding restriction fragment of clone comprising correct 1Kbp sequences Gel cuts, and using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) according to the guidance of manufacturer Carry out purification.Then by the fragment and the pDAB7471 (positions for having used restriction enzyme Spe I digestion and subsequent dephosphorylized purification Put -1 plasmid backbone) (Figure 70) or pDAB7451 (plasmid backbone of position -2) (Figure 71) in coupled reaction combine.In following bar Implement connection under part：1: 5 carrier in the reaction volume of 20 μ l: insert ratio and 500 unit T4DNA ligases (Invitrogen Life Technologies, Carlsbad, CA), under conditions of incubating 16 hours in 16 DEG C of water-baths.With 5 μ l coupled reactions are converted to 50 μ l escherichia coli MAX afterwards DH5α^TMCompetent cell In (Invitrogen Life Technologies, Carlsbad, CA), and the bed board under alternative condition described in manufacturer.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml chloromycetin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 units SpeI (New England Biolabs, Beverly, MA) digest the detached plasmid DNA of 3 μ g, and in 37 DEG C of incubations 1 hour.In 100V to restricted DNA electrophoresis 1 hour in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide. Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to 1.0Kbp and 4.96Kbp The presence of (pDAB7471 carriers) or 1.0Kbp and about 5.0Kbp (pDAB7451 carriers) DNA fragmentation is judging expected plasmid gram It is grand.The plasmid of gained is respectively designated as pDAB7423 (the non-autonomous donor in position -1), and (Figure 76) (position -2 is non-certainly with pDAB7454 Main donor) (Figure 77).

H. position 1ZFN+HR donor sequences：Combination plasmid

(such as one plasmid another bag comprising ZFN elements is delivered in plant cell as two separate plasmids Donor sequences containing herbicide tolerant) alternative strategy, single plasmid be transformed into comprising in this patent illustrate be necessary Element.Plasmid is combined described in the present embodiment comprising being designed for the specific IPP2K locus of targeting and there to produce double-strand disconnected The ZFN for splitting and the autonomous PAT PTU for being designed for being integrated in those broken sites and/or non-autonomous 2A/PAT PTU and confession Both side wings.

Utilize(it uses locus specificity restructuring (Landy, the A. (1989) based on bacteriophage lambda to technology Ann.Rev.Biochem.58：913)) by carrier pDAB7422 and pDAB7423 (described in embodiment 6E and 6G) transformation IntoPurpose carrier.Once being converted, easily (will can be housed in comprising ZFN expression cassettesInto In carrier (entry vector)) plasmid mobilize (mobilize) to purpose carrier, create ZFN/ donor combination plasmids.37 DEG C the such plasmids of each 1 μ g are digested up to 1 hour with 10 unit Not I (New England Biolabs, Beverly, MA). 65 DEG C, by Not I restriction endonucleases heat inactivation 15 minutes, subsequently use 3 unit shrimp alkaline phosphotases (SAP) (Roche Diagnostics GmbH, Mannheim, Germany) carried out dephosphorylation at 37 DEG C up to 1 hour to fragment ends. In 100V to restricted DNA electrophoresis 1 hour in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide.With ultraviolet Line manifests carrier segments (pDB7422=7.317Kbp, pDAB7423=5.971Kbp), is estimated by comparing with 1Kbp DNA ladders Meter size, carries out gel cutting, subsequently using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) according to Guidance according to manufacturer carries out purification.

Then by the carrier segments with includeTechnology element attR1, ccdB, Cm^RAnd attR2 2.274Kbp Not I fragments combine in the coupled reaction implemented under the conditions of following：1: 5 in the reaction volume of 20 μ l Carrier: insert ratio and 500 unit T4DNA lipases (InvitrogenLife Technologies, Carlsbad, CA), under conditions of incubating 16 hours in 16 DEG C of water-baths.Subsequently the coupled reaction of 5 μ l is converted to 50 μ l escherichia coli One ccdB Survival^TMCompetent cell (Invitrogen Life Technologies, Carlsbad, CA) In, and the bed board under alternative condition described in manufacturer.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml chloromycetin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000x g.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 units EcoRII (New EnglandBioLabs, Inc., Beverly, MA) digest the detached plasmid DNA of 3 μ g, and 37 DEG C incubate 1 hour.In 100V to restricted DNA electrophoresis 1 in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Hour.Manifest fragment with ultraviolet, and estimate clip size by comparing with 1Kbp DNA ladders.According to 1.448Kbp, The DNA fragmentation (autonomous PAT PTU positions -1HR donors) of 1.946Kbp and 6.197Kbp and 5.807Kbp's and 2.438Kbp The presence of DNA fragmentation (non-autonomous PAT positions -1HR donors) is judging expected plasmid cloning.The plasmid of gained is respectively designated as PDAB7424 (JingThe autonomous donor in position -1 of reorganization) (Figure 78) and pDAB7425 (JingReorganization The non-autonomous donor in position -1) (Figure 79).

As the result of these clone operations, plasmid pDAB7424 and pDAB7425 are designed asPurpose is carried Body.PDAB7412 has as including following elementInto the feature of carrier：ZmUbi1v.Z/ZFN12/ Zm Per5 3’UTR.In order to by ZFN expression cassettes (Into carrier) it is transferred to autonomous or non-autonomous donor point Son (Purpose carrier) in, with 50ng (entering carrier)：The ratio of 150ng/ μ l (purpose carrier) implements LR Clonase^TMII (Invitrogen Life Technologies, Carlsbad, CA) reacts, summarized such as manufacturer that Sample is carried out.The positive combination carrier of gained be named as pDAB7426 (the autonomous HR donors/ZFN12 in position -1) (Figure 80) and PDAB7427 (non-autonomous HR donors/ZFN12) is (Figure 81).

Embodiment 19：ZFN and donor dna are to the delivery in plant cell

In order to realize ZFN is mediated, donor dna to the integration in Plant Genome via targeted integration, it should be understood that It is that needs deliver the DNA of coding ZFN, then the expression function ZFN albumen in plant cell.What is also needed is donor dna to The associated delivery of the plant cell so that feature ZFN albumen can induce double-strand break on target DNA, then the double-strand Rupture to be driven to the homology in target gene seat via donor dna and integrate and repaired.Skilled in the art realises that, can lead to Cross several methods to realize the expression of feature ZFN albumen, the including but not limited to gene transfer or ZFN of ZFN encoding constructs The transient expression of encoding constructs.In both of these case, realize simultaneously in plant cell feature ZFN albumen expression and The delivery of donor dna is driving targeted integration.

In the embodiment quoted here, we are demonstrated for delivering ZFN coding DNAs and donor together in plant cell The method of DNA.Those skilled in the art can using any one of various DNA delivering methods for being suitable to plant cell, including but not It is limited to agrobacterium-mediated conversion, the DNA based on Biolistic to deliver or Whiskers^TMThe DNA of mediation is delivered.Institute here In the embodiment stated, combine to implement Whiskers using donor dna with the various of ZFN encoding DNA constructs^TMMediation DNA deliver experiment.These combinations include single plasmid 1) comprising both ZFN coded sequences and donor dna and 2) two not Same plasmid, one comprising ZFN coded sequences, another includes donor dna.In another embodiment, using donor DNA is delivered with the various DNA combined to implement based on Biolistic of ZFN encoding DNA constructs.Skilled in the art realises that 1) these combinations may include comprising ZFN coded sequences and the single plasmid of both donor dnas and 2) two different plasmids, one Comprising ZFN coded sequences, another includes donor dna.

A.Whiskers ^TM The DNA of mediation is delivered

Such as described earlier herein, there is Hi-II cell cultures in the embryo for generating Semen Maydiss, and as the work of confirmation targeted integration The source of plant cell.Those skilled in the art are using from cell culture derived from various plants species or from many plantations Source of the derivative differentiation plant tissue of thing species as the live plant cells for confirming targeted integration.

In the present embodiment, 12ml is passed from the PCV of previous cryopreservation cell line plus 28ml conditioned culture medias 80ml GN6 fluid mediums in culture to 500ml Erlenmeyer flasks (N6 culture medium (Chu etc., 1975), 2.0mg/L 2,4-D, 30g/L sucrose, pH5.8) in, and place on bottle swingging machine (125rpm, 28 DEG C).Using identical cell System repeats the step twice so that altogether 36mlPCV distributes between 3 flasks.After 24 hours, GN6 fluid mediums are removed, and With 72ml GN6S/M osmotic mediums (N6 culture medium, 2.0mg/L 2,4-D, 30g/L sucrose, 45.5g/L Sorbitol, 45.5g/ L Mannitol, 100mg/L muscle-inositol, pH6.0) replace.It is warm in the dark at 28 DEG C in moderate agitation (125rpm) situation Flask is educated up to 30-35 minutes.During incubation period, by the way that 8.1ml GN6S/M fluid mediums are added to 405mg is aseptic, carbon SiClx whisker (Advanced Composite Materials, LLC, Greer, SC) is outstanding to prepare 50mg/ml silicon carbide whiskers Supernatant liquid.

After incubating in GN6S/M osmotic mediums, the inclusions of each flask are incorporated in 250ml centrifuge bottles.Burning All cell settlements in bottle draw the inclusions volume more than about 14ml GN6S/M liquid to bottom, and collect aseptic 1L flasks in, for future usage.At full throttle make the whisker suspension vortex mixed of advance moistening 60 seconds, be then added to Centrifuge bottle.

In the example that will be delivered to plus the single plasmid of both donor dnas comprising ZFN coded sequences in plant cell In son, the cyclic plasmid DNA of 170 μ g purification is added into bottle.Two different plasmids are carried out into common delivery, (one includes ZFN coded sequences and another include donor dna) an alternative example in, assess with regard to amount of DNA various strategies.It is a kind of Strategy is using 85 μ g donor dnas and 85 μ g zinc finger coding DNAs.Other improved forms are using 10,5 or 1 times of donor dnas than 1 times of zinc Refer to the molar ratio of DNA, this is based on the individual size of plasmid (in terms of thousand base pairs) so that each bottle adds 170 μ altogether g DNA.In the case of all common deliveries, DNA is pre-cooled in pipe, added afterwards into centrifuge bottle.Once addition DNA, Immediately bottle is placed on into improvement the commercial paint mixer of Red Devil 5400 (Red Devil Equipment Co., Plymouth, MN) in, and stir 10 seconds.It is after agitation, the mixture of cell, culture medium, whisker and DNA is fresh together with 125ml GN6 fluid mediums add the inclusions to 1L flasks together to reduce osmoticum (osmoticant).Permissive cell is setting It is set on the bottle swingging machine of 125rpm and recovers 2 hours.Using the glass cell harvestor unit being connected with house vacuum pipeline by 6mL Scattered suspension is filtered to Whatman#4 filter paper (5.5cm) so that each bottle obtains 60 filter paper.Filter paper is placed In 60x20mm GN6 solid mediums (identical with GN6 fluid mediums, simply with 2.5g/L Gelrite gellant) flat board On, and cultivate 1 week under dark condition at 28 DEG C.

B. the DNA of Biolistic mediation is delivered

In the example quoted here, be there is into suspension Secondary Culture in the embryo of Semen Maydiss little in GN6 fluid mediums 24 When, experiment described earlier herein is carried out afterwards.Excessive fluid medium is taken out, and about 0.4PCV cells are being contained into useful Diameter 2.5cm is sparsely coated in the heart in the 100x15mm Pi Shi wares of the GN6S/M culture medium of 2.5g/L Gelrite solidifications A circle.Cell is cultivated 4 hours under dark condition.In order to be coated with Biolistic granule with DNA, by 1.0 microns of 3mg The gold grain of diameter 100% ethanol purge 1 time, is cleaned 2 times with sterile distilled water, and is managed in silication Eppendorf in 50 μ l In water in it is resuspended.Will altogether 5 μ g plasmid DNA, 20 μ l spermidines (0.1M) and 50 μ l calcium chloride (2.5M) be added separately to gold Suspension, and vortex mixed.By mixture in incubation at room temperature 10 minutes, with 10,000rpm precipitations in small table centrifuge It is 10 seconds, resuspended in 100% 60 μ l cold ethanol, and 8-9 μ l are distributed to each grand carrier.

Using Biolistic PDS-1000/He^TMSystem (Bio-Rad Laboratories, Hercules, CA) carries out banging Hit.The flat board containing cell is placed under the conditions of 1100psi and 27 inches Hg vacuums on the support of centre, and follows operation Handbook is being bombarded.16 hours after bombardment, tissue is transferred to into GN6 (1H) culture medium with fritter, and at 28 DEG C in dark bar 2-3 is cultivated under part all.Continue in week to shift per 2-4, until the presumption transgenic for occurring being integrated from donor dna separates group.Via Biolistic mediation DNA deliver produce presumption donor dna integrate event identification, separate and regenerate be used for via Whiskers^TMThe method that the DNA of mediation delivers the presumption donor dna integration event for producing is identical, and is described below.

C. being identified and isolated from for targeted integration transgenic event is estimated

1 week after DNA deliveries, filter paper is transferred to into 60x20mm GN6 (1H) Selective agar medium (N6 culture medium, 2.0mg/L 2,4-D, 30g/L sucrose, 100mg/L muscle-inositol, 1.0mg/L is from Herbiace's (Meiji Seika, Japan) Bialaphos, 2.5g/L Gelrite, pH5.8) on flat board.These selection flat boards are incubated 1 week in the dark at 28 DEG C.

After selecting 1 week in the dark, 37- is maintained at into equipped with 3.0mL by the way that half is scraped from the cell of every piece of flat board 38 DEG C of GN6 agarose medias (N6 culture medium, 2.0mg/L 2,4-D, 30g/L sucrose, 100mg/L muscle-inositol, 7g/LAgarose, pH5.8, in 121 DEG C of autoclavings only 10 minutes) and 1mg/L from Herbiace's Tissue is embedded on fresh culture in the pipe of Bialaphos.

Agarose/tissue mixture is smashed with spatula, subsequently equably pours into 3mL agaroses/tissue mixture On the surface of Pi Shi wares of the 100x15mm comprising GN6 (1H) culture medium.The process is repeated to both sides half block in every piece of flat board.Once Embedding institute in a organized way, then usesOrEach seal plate, and at 28 DEG C under dark condition Culture is up to 10 weeks.The presumption conversion for growing in the selection conditions is taken out from embedding flat board and separates group, and be transferred to Fresh selection medium in 60x20mm flat boards.If continued propagation after about 2 weeks be it will be evident that if event be considered as to being applied Herbicide (Bialophos) it is resistant, subsequently the aliquot of cell is harvested into 2mL Eppendorf pipes, for base Because of type analysis.

Those skilled in the art can utilize the gene that any appropriately selected mark is encoded in donor dna, and should to living cells With suitable alternative condition.For example, can perform alternative selected marker gene (such as AAD-1, such as WO 2005/107437A2 institutes State) as donor, for integrating the selection and recovery of event in maize cell described herein.

Embodiment 20：Targeted integration events are screened via pcr gene type typing

In the present embodiment, pcr gene type typing is interpreted as including but not limited to polymerase chain reaction (PCR) and is amplified from Genomic DNA derived from detached Semen Maydiss corpus callosum (its prediction includes the donor dna in embedded genome), is followed by PCR expansions The standard Cloned culturing of volume increase thing.The method of pcr gene type typing has been documented (such as Rios, G. etc. (2002)Plant J.32：243-253), and can be applicable to from any plant species or organization type (including cell culture) Derivative genomic DNA.

Those skilled in the art may be configured to the strategy of pcr gene type typing, and it includes but is not limited to plant gene In group in the amplification of particular sequence, Plant Genome in the amplification of multiple particular sequences, Plant Genome nonspecific sequence expansion Increase or its combination.Amplification can be cloned and be sequenced (as set forth in the present embodiment), or the direct sequence of amplified production Row analysis.Skilled in the art realises that can be used to analyze the alternative approach of produced amplified fragments herein.

In an embodiment as herein described, in PCR amplifications the antisense oligonucleotide primer special to gene target is adopted Thing.In another embodiment as herein described, in PCR amplifications the antisense oligonucleotide primer special to donor DNA sequences is adopted Thing.Another embodiment includes the combination of the oligonucleotide primers for combining both gene target sequence and donor DNA sequences.Ability Field technique personnel may be configured to inquire other primer combination of genome and amplified reaction.

A. extracting genome DNA

Genomic DNA (gDNA) is extracted from maize cell described in detached, herbicide tolerant embodiment 19, and is used as The template of pcr gene type typing assay.According to96 botanical agents boxes (QIAGEN Inc., Valencia, CA) are in detail The manufacturer's scheme stated enriches the herbicide tolerant detached as described above of cell volume (PCV) from about 100-300 μ l HiII corpus callosum extracts gDNA.The eluting genomic DNA in the elution buffer that 100 μ l test kits are provided, produces 20-200ng/ The final concentration of μ l, subsequently via following summary PCR-based genotyping method analyzing.

B. it is used for the design of primers of pcr gene type typing

Those skilled in the art can be using various strategies for designing and implementing the genotyping of PCR-based.Design It is feasible for being annealed to gene target, donor DNA sequences and/or both oligonucleotide primers of combination.In order to design energy It is annealed to not for the widow of the IPP2K gene targets in the homology flank area encompassed that is fabricated into donor DNA molecule Nucleotide primer, the plasmid cloning comprising other gene target sequence data is characterized via DNA sequencing.Using CEQ^TMDTCS- is fast Speed starts test kit (Beckman-Coulter, Palo Alto, CA) carries out the double-strand sequencing of plasmid cloning as described in manufacturer Reaction.Using Performa DTR gel filtration cylinders (Edge BioSystems, Gaithersburg, MD) such as manufacturer's scheme Purification reaction as described.In Beckman-Coulter CEQ^TMAnalytical sequence reaction in 2000XL DNA analysis systems, and Using Sequencher^TM4.1.4 version (Gene Codes Corporation, Ann Arbor, MI) carries out nucleotide sign.This A little sequences are depicted in Figure 91 (SEQ corresponding to ZFN targeted areas upstream (5 ' -) and the IPP2K gene regions of downstream (3 ' -) ID NO：141) with Figure 92 (SEQ IDNO：142) in.

In the example for proposing here, by Integrated DNA Technologies, Inc. (Coralville, IA) Synthesize all of oligonucleotide primers under the conditions of standard desalination, and be diluted with water to 100 μM of concentration.The following group forward and reverse is few Nucleotide primer is designed for being annealed to gDNA sequences outside the border of donor DNA sequences, that IPP2K gene targets are special Row.These oligonucleotide are as follows：

5’-TGGACGGAGCGAGAGCCAGAATTCGACGCT G-3’(SEQ ID NO：153)

5’-GTGCAAGAATGTATTGGGAATCAACCTGAT G-3’(SEQ ID NO：154).

Outside the border that second group of forward and reverse oligonucleotide primers is also designed for being annealed to donor DNA sequences, The special gDNA sequences of IPP2K gene targets, but it is nested within first pair：

5’-CTGTGGTACCAGTACTAGTACCAGCATC-3’(SEQ ID NO：155)

5’-TCT TGGATCAAGGCATCAAGC ATTCCAATCT-3’(SEQ ID NO：156).

Be additionally designed for specificity be annealed to donor dna corresponding with the coding region of herbicide tolerance genes forward direction and Reverse oligonucleotide primer：

5’-TGGGTAACTGGCCTAACTGG-3’(SEQ ID NO：157)

5’-TGGAAGGCTAGGAACGCTTA-3’(SEQ ID NO：158)

5’-CCAGTTAGGCCAGTTACCCA-3’(SEQ ID NO：159)

5’TAAGCGTTCCTAGCCTTCCA-3’(SEQ ID NO：160).

C. the special PCR amplifications of donor dna

Using by TaKaRa Biotechnology Inc., Seta 3-4-1, Otsu, Shiga, 520-2193, Japan The reagent of offer implements primary PCR amplification reaction, and its composition is as follows：2.5μl 10X Ex TaqPCR^TMBuffer, 40-200ng Double stranded genomic dna template, 10 μM of positive oligonucleotide primers, 10 μM of reverse oligonucleotide primers, 2 μ l dNTP mixture are (each 2.5mM)、16μl H₂O, 0.5 μ l (2.5 units) Ex Taq^TMArchaeal dna polymerase.Using Bio-Rad, 96 sample DNAs Engine Tetrad2, Peltier thermal cycler (Hercules, CA) enter performing PCR reaction under following cycling condition：94℃3 Minute/1 circulation；94 DEG C 30 seconds, 64 DEG C 30 seconds, 72 DEG C 5 minutes/35 circulation；72 DEG C of 10 minutes/1 circulations；4 DEG C/protect Hold.

Subsequently expand the amplified production of first PCR reactions again in the reactions of the PCR again comprising following composition：2.5μl 10X Ex Taq PCR^TM(first PCR is reacted in H for buffer, 2 μ l templates₂1: 100 dilution in O), 10 μM of positive antisense oligonucleotide primers Thing, 10 μM of reverse oligonucleotide primers, 2 μ l dNTP mixture (each 2.5mM), 16 μ l H₂O, 0.5 μ l (2.5 units) Ex Taq^TMArchaeal dna polymerase.Using Bio-Rad, 96 sample DNA Engine Tetrad2, Peltier thermal cyclers (Hercules, CA performing PCR reaction) is entered under following cycling condition：95 DEG C of 1 minute/1 circulations；94 DEG C 15 seconds, 61 DEG C 30 seconds, 72 DEG C 30 seconds/ 30 circulations；72 DEG C of 1 minute/1 circulations；4 DEG C/keep.It is being supplemented with the 1.0%TAE agarose gel of 0.5% ethidium bromide In in 100V to each amplified production electrophoresis of 10 μ l 1 hour.Manifest amplified fragments with ultraviolet, and by with 1Kbp Plus DNA Terraced (Invitrogen Life Technologies, Carlsbad, CA) compares to estimate clip size.As shown in Figure 82, root The PCR primer comprising expected fragment is judged according to the presence of 0.317Kbp DNA fragmentations.

Embodiment 21：The detection of targeted integration events

In the herbicide tolerant event of the integration donor DNA molecule comprising encoding herbicide-tolerant box gene, a definite proportion The event of example is the product that donor dna targeted integration enters in the double-strand break site of ZFN inductions.In order to distinguish these targeting Integration event with those from event derived from herbicide tolerance genes box random integration, using the genotyping plan of PCR-based Slightly, it uses the combination of the PCR primer special plus donor of the PCR primer of genome specific and subsequent genome specific.

A. the special amplification of the amplification of genome specific and subsequent genome/donor

In the present embodiment, first PCR reactions are special using donor integration upstream and downstream IPP2K gene targets area of area Oligonucleotide primers (such as Figure 92 and 93).Using by TaKaRa BiotechnologyInc., Seta 3-4-1, Otsu, The reagent that Shiga, 520-2193, Japan are provided implements primary PCR amplification reaction, and its composition is as follows：2.5μl10X Ex Taq PCR^TMBuffer, 40-200ng double-strand Semen Maydiss gDNA templates, 10 μM of positive oligonucleotide primers, 10 μM of reverse oligonucleotides draw Thing, 2 μ l dNTP mixture (each 2.5mM), 16 μ l H₂O, 0.5 μ l (2.5 units) Ex Taq^TMArchaeal dna polymerase.Use Bio-Rad, 96 sample DNA Engine Tetrad2, Peltier thermal cyclers (Hercules, CA) are under following cycling condition Enter performing PCR reaction：94 DEG C of 3 minutes/1 circulations；94 DEG C 30 seconds, 64 DEG C 30 seconds, 72 DEG C 5 minutes/35 circulation；72 DEG C 10 points Circulate clock/1；4 DEG C/keep.

Subsequently by first PCR product in H₂1: 100 dilution in O, and as the mould of two different reactions of PCR again Plate DNA.In the present embodiment, again reaction, using the primer combined in IPP2K genomic regions and donor molecule, is produced horizontal The amplicon on the integration border between genome and donor.First is reacted the 5 ' sides focused between genome and donor Boundary.Second is reacted the 3 ' borders focused between donor and genome.Two reactions constitute as follows：2.5μl 10X Ex Taq PCR^TMBuffer, 2 μ l templates [first PCR reactants 1: 100 dilute], 10 μM of positive oligonucleotide primers, 10 μM reversely Oligonucleotide primers, 2 μ l dNTP mixture (each 2.5mM), 16 μ l H2O, 0.5 μ l (2.5 units) Ex Taq^TMDNA is polymerized Enzyme.Using Bio-Rad, 96 sample DNA Engine Tetrad2, Peltier thermal cyclers (Hercules, CA) are being followed as follows Enter performing PCR reaction under the conditions of ring：94 DEG C of 3 minutes/1 circulations；94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 2 minutes/35 circulation；72 DEG C of 10 minutes/1 circulation；4 DEG C/keep.At 100V pair in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Each PCR reactants electrophoresis 1 hour again of 20 μ l.

Manifest amplified fragments with ultraviolet, and by with 1Kbp Plus DNA ladder (Invitrogen LifeTechnologies, Carlsbad, CA) compare to estimate clip size.According to DNA fragmentation 1.65Kbp (5 ' border) (figures Or 1.99Kbp (3 ' border) presence (Figure 84) is come PCR derived from the targeted integration in judging from donor to IPP2K genes 83) Product.These fragments are carried out with gel cutting, and using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) purification is carried out according to the guidance of manufacturer.Subsequently use TOPO TATest kit (contains2.1 carry Body) andTOP10 Competent Bacillus coli cells (Invitrogen Life Technologies, Carlsbad, CA) fragment of purification is cloned in ApCR2.1 plasmids according to the scheme of manufacturer.

Individual colonies are seeded to into the Falcon pipe (Becton- that 14ml is supplemented with the TB of 50 μ l/ml kanamycin containing 2ml Dickinson, Franklin Lakes, NJ) in, and incubate 16 hours at 37 DEG C in the case where being shaken with 200rpm.Incubate Afterwards, 1.5ml cells are transferred in 1.7ml Costar microcentrifugal tubes (FisherScientific, Pittsburgh, PA), And precipitated 1 minute with 16,000xg.Supernatant is removed, and is usedPlasmid kit (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) isolated plasmid dna as above.With 10 units EcoRI (New England BioLabs, Beverly, MA) digest the detached plasmids of 3 μ g.By all plasmid digests Thing is incubated 1 hour at 37 DEG C.In 100V to restricted in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide DNA electrophoresis 1 hour.Manifest fragment with ultraviolet, and by with 1Kbp Plus DNA ladder (Invitrogen LifeTechnologies, Carlsbad, CA) compare to estimate clip size.

According to 3.9KbpThe presence of the insertion DNA fragmentation of the suitable size outside 2.1 carriers is expected to judge Plasmid cloning.Using CEQ^TMDTCS- quickly starts test kit (Beckman-Coulter, PaloAlto, CA) such as institute of manufacturer Implement the double-strand sequencing reaction of plasmid cloning as stating.Using PerformaDTR gel filtration cylinders (Edge BioSystems, Gaithersburg, MD) purification reaction as described in the scheme of manufacturer.In Beckman-Coulter CEQ^TM Analytical sequence reaction in 2000XL DNA analysis systems, and use Sequencher^TM4.1.4 version (Gene Codes Corporation, Ann Arbor, MI) carrying out nucleotide sign.Using 10.1 editions (Invitrogen of Vector NTi Life Technologies, Carlsbad, CA) carry out nucleotide comparison.

It is carried out as follows the sequence data analysis from targeted integration events (event #073).To integrating across whole gene group The first PCR primer in site is focused the amplification again on 5 ' or 3 ' borders between genome and donor.With these again The corresponding cloned sequence of amplified production is compared with the expected sequence of wild type IPP2K genome sequences and targeted integration events Clearly indicate the precise integration that donor dna is there occurs in target site.

The nucleotide sequence of IPP2K genomic locus, genome/donor border are corresponding with IPP2K homology flanks The nucleotide sequence of donor areas and the nucleotide sequence of herbicide tolerant box are maintained in from multiple clones derived from the event In PCR primer.Therefore, the event represents the homology driving that the double-strand break that ZFN is mediated occurs at specific gene target and repaiies The genome of the targeted integration of multiple and donor dna.Have been obtained for representing other transformation event that unique targeted integration occurs, card Real method teaching herein is reproducible in Semen Maydiss corpus callosum.Those skilled in the art can be applied to these methods appoint What think to want any gene target of targeted integration in plant species.

B. nested type genome/special amplification of donor

In the present embodiment, it is first and subsequently PCR reaction both of which again using donor integrate region upstream or The special oligonucleotide primers (annex V and VI) in the IPP2K gene targets region in downstream and the special oligonucleotide of donor sequences The combination of primer.In the present example, use by TaKaRaBiotechnology Inc., Seta 3-4-1, Otsu, Shiga, The reagent that 520-2193, Japan are provided implements primary PCR amplification reaction, and its composition is as follows：2.5μl 10X Ex Taq PCR^TM Buffer, 40-200ng double-strand Semen Maydiss gDNA templates, 10 μM of positive oligonucleotide primers, 10 μM of reverse oligonucleotide primers, 2 μ LdNTP mixture (each 2.5mM), 16 μ l H₂O, 0.5 μ l (2.5 units) Ex Taq^TMArchaeal dna polymerase.Using Bio-Rad, 96 sample DNA Engine Tetrad2, Peltier thermal cyclers (Hercules, CA) enter performing PCR under following cycling condition Reaction：94 DEG C of 3 minutes/1 circulations；94 DEG C 30 seconds 30 seconds, 52 DEG C or 64 DEG C, 72 DEG C 2 minutes/35 circulation；72 DEG C 10 minutes/ 1 circulation；4 DEG C/keep.

Then first PCR is reacted in H₂1: 100 is diluted in O, and as the template DNA of the reactions of PCR again.In this enforcement In scheme, then secondary response also utilizes the primer combined in IPP2K genome areas and donor molecule, produce across genome and The amplicon on the integration border between donor.The concrete primer for being used determines that amplicon is focused between genome and donor 5 ' or 3 ' borders.The reagent composition of these reactions is as follows：2.5μl 10X Ex Taq PCR^TMBuffer, 2 μ l templates are [just Secondary PCR reaction 1: 100 dilutes], 10 μM of positive oligonucleotide primers, 10 μM of reverse oligonucleotide primers, 2 μ l dNTP mixture (each 2.5mM), 16 μ l H₂O, 0.5 μ l (2.5 units) Ex Taq^TMArchaeal dna polymerase.Using Bio-Rad, 96 sample DNAs Engine Tetrad2, Peltier thermal cycler (Hercules, CA) enter performing PCR reaction under following cycling condition：94℃3 Minute/1 circulation；94 DEG C 30 seconds 30 seconds, 54 DEG C or 60 DEG C, 72 DEG C 2 minutes/35 circulation；72 DEG C of 10 minutes/1 circulations；4 DEG C/keep.In the 100V reactions of PCR again each to 20 μ l in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide Electrophoresis 1 hour.

Manifest amplified fragments with ultraviolet, and by with 1Kbp Plus DNA ladder (Invitrogen LifeTechnologies, Carlsbad, CA) compare to estimate clip size.According to DNA fragmentation 1.35Kbp (5 ' border) (figures Or 1.66Kbp (3 ' border) presence (Figure 86) is come PCR derived from the targeted integration in judging from donor to IPP2K genes 85) Product.These fragments are carried out with gel cutting, and using QIA quick gel extraction kits (QIAGEN Inc., Valencia, CA) purification is carried out according to the guidance of manufacturer.Then TOPO TA are usedTest kit (contains2.1 carry Body) and OneTOP10 Competent Bacillus coli cells (Invitrogen Life Technologies, Carlsbad, CA) fragment of purification is cloned in pCR2.1 plasmids according to the scheme of manufacturer.

C. nucleotide sequence analysises of genotyping PCR primer

Individual colonies described in embodiment 21B are seeded to into the TB that 14ml is supplemented with containing 2ml 50 μ l/ml kanamycin In Falcon pipes (Becton-Dickinson, Franklin Lakes, NJ), and in the case where being shaken with 200rpm at 37 DEG C Incubate 16 hours.After incubation, by 1.5ml cells be transferred to 1.7ml Costar microcentrifugal tubes (Fisher Scientific, Pittsburgh, PA) in, and precipitated 1 minute with 16,000x g.Supernatant is removed, and is usedPlasmid is tried Agent box (BD Biosciences/Clontech/Macherey-Nagel, Palo Alto, CA) separates as above matter Grain DNA.The detached plasmids of 3 μ g are digested with 10 units EcoRI (New England BioLabs, Beverly, MA).Will be all Plasmid digestions 37 DEG C incubate 1 hour.In 100V in the 1.0%TAE agarose gel for be supplemented with 0.5% ethidium bromide To restricted DNA electrophoresis 1 hour.Manifest fragment with ultraviolet, and by with 1Kbp Plus DNA ladders (Invitrogen Life Technologies, Carlsbad, CA) compare to estimate clip size.

According to 3.9KbpThe presence of the insertion DNA fragmentation outside 2.1 carriers is judging plasmid cloning.Using CEQ^TM DTCS- quickly starts test kit (Beckman-Coulter, Palo Alto, CA) and implements plasmid gram as described in manufacturer Grand double-strand sequencing reaction.Using Performa DTR gel filtration cylinders (Edge BioSystems, Gaithersburg, MD) Purification reaction as described in the scheme of manufacturer.In Beckman-Coulter CEQ^TMIn 2000XL DNA analysis systems Analytical sequence is reacted, and uses Sequencher^TM4.1.4 version (Gene Codes Corporation, Ann Arbor, MI) is come Carry out nucleotide sign.Using Vector NTi 10.1 editions (Invitrogen Life Technologies, Carlsbad, CA) nucleotide comparison is carried out.

Also obtain cover from derived from multiple targeted integration events in upstream (5 ' -) IPP2K genome sequences and donor dna Between border sequence data, including cover from derived from multiple targeted integration events in donor dna and downstream (3 ' -) The sequence data on the border between IPP2K genome sequences and including from single conversion corpus callosum event (#114) it is derivative on The sequence data of trip (5 ' -) border sequence.The targeted integration events (#114) of conversion are that autonomous donor is integrated into IPP2K gene targets Result in thing.

In these analyses, both pcr amplification reaction first and again is focused between genome and donor 5 ' or 3 ' borders.With these again the corresponding cloned sequence of amplified production it is whole with wild type IPP2K genome sequences and targeting The comparison of the expected sequence of conjunction event is disclosed in the integration that target site occurs donor dna.The nucleotide of IPP2K genomic locus The nucleotide sequence and herbicide tolerant of sequence, genome/donor border donor areas corresponding with IPP2K homology flanks The nucleotide sequence of box is maintained in from multiple clone PCR products derived from the event.

Therefore, the event is represented and there occurs that the homology driving of the double-strand break that ZFN is mediated is repaiied at specific gene target Multiple genome.Have been obtained for representing other transformation event that unique targeted integration occurs, it was demonstrated that method teaching herein It is reproducible in Semen Maydiss corpus callosum.Those skilled in the art can be applied to these methods think to think in any plant species Want any gene target of targeted integration.

Embodiment 22：From the whole plant that Semen Maydiss callus tissue Regenerated energy is educated

Can by from derived from HiII cell cultures, the detached corpus callosum of the maize cell of herbicide tolerant has been regenerated as Milpa that is whole, can educating.Those skilled in the art can from many embryos occur corn cell culture regeneration it is complete, can educate Milpa.

In the present embodiment, by the way that detached callus tissue is transferred to based on the inducing culture 28 of the basic element of cell division (1H) starting the callosal regeneration of detached, Bialophos resistances HiII, the inducing culture based on the basic element of cell division Base 28 (1H) includes MS salts and vitamin, 30.0g/L sucrose, 5mg/L benayl aminopurines, 0.25mg/L 2,4-D, 1mg/L Bialaphos and 2.5g/L Gelrite；pH5.7.Permissive cell growth 1 week in low illumination (13 μ Em-2s-1), then turns High light is moved to according to condition (40 μ Em-2s-1) up to 1 week.Then cell is transferred to into regeneration culture medium 36 (1H), itself and induction are trained Foster base is identical, and simply it lacks plant growth regulator.By hand instrument cuts out little (3-5cm) plantlet, and is put into and includes SHGA culture medium (Schenk and Hildebrandt basis salts and vitamin, 1972, Can.J.Bot 50：199-204；1g/L Muscle-inositol, 10g/L sucrose, 2.0g/L Gelrite, pH5.8) aseptic 150x 25-mm Glass Culture Tubes in.

Once plantlet development is sufficiently large and differentiation root system and shoot system, they are implanted into equipped with Metro- In 4 inches of tanks of the growth mediums of Mix 360 (Sun Gro Horticulture Canada Ltd.), and it is placed on greenhouse In.Plantlet is covered wholly or in part up to 2-7 days with transparent plastic cup, is then migrated to equipped with by 95%Metro-Mix 5 gallon cans of the mixture of 360 growth mediums and 5% clay/loam composition, and cultivate to maturation.Plant can be carried out certainly Flower pollination carries out cross pollination to produce T1 or F1 seeds respectively with inbred line.Those skilled in the art can be to the plant of regeneration Carry out self-pollination or carry out cross pollination to realize corn breeding with various germplasm by the plant of regeneration.

Can be in U.S. Patent Application Publication text US-2003-0232410；US-2005-0026157；US-2005- 0064474；US-2005-0208489 and US-2006-0188987；The U.S. Patent application stream submitted to on July 26th, 2006 Water number 11/493, finds the other information related to targeting cutting, targeting restructuring and targeted integration in 423, complete by referring to The whole disclosure for including these applications is for all purposes.

All patents mentioned herein, patent application and publication are completely included by referring to for institute with this Purposefully.

Although in order to understand that clearly purpose is illustrated with more detail providing disclosure, to this Art personnel are it is readily apparent that various changes can be implemented on the premise of the spirit or scope without departing substantially from present disclosure Change and modify.Thus, description above and embodiment are not construed as limiting.

Sequence table

<110>Dow AgroSciences (DOW AGROSCIENCES LLC)

Sangamo Biosciences Inc. (SANGAMO BIOSCIENCES, INC.)

<120>Through the non-standard zinc finger protein for optimizing

<130>8325-4002.40

<140>PCT/US2007/025455

<141>2007-12-13

<150>60/874,911

<151>2006-12-14

<150>60/932,497

<151>2007-05-30

<160>199

<170>PatentIn version 3.5

<210>1

<211>25

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<220>

<221>MISC_FEATURE

<222>(2)..(3)

<223>Xaa=any one aminoacid

<220>

<221>MISC_FEATURE

<222>(4)..(5)

<223>Xaa=any one aminoacid

Xaa can be presence or absence of

<220>

<221>MISC_FEATURE

<222>(7)..(18)

<223>Xaa=any one aminoacid

<220>

<221>MISC_FEATURE

<222>(20)..(22)

<223>Xaa=any one aminoacid

<220>

<221>MISC_FEATURE

<222>(23)..(24)

<223>Xaa=any one aminoacid

Xaa can be existed or non-existent

<400>1

Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa His Xaa Xaa Xaa Xaa Xaa His

20 25

<210>2

<211>6

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223>Xaa=any one aminoacid (preferred A, K or T)

<220>

<221>MISC_FEATURE

<222>(3)..(3)

<223>Xaa=any one aminoacid (preferred Q, E or R)

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223>Xaa=any one aminoacid (preferred G)

<400>2

His Xaa Xaa Arg Cys Xaa

1 5

<210>3

<211>35

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<220>

<221>MISC_FEATURE

<222>(2)..(3)

<223>Xaa=any one aminoacid

<220>

<221>MISC_FEATURE

<222>(4)..(5)

<223>Xaa=any one aminoacid

Xaa can be existed or non-existent

<220>

<221>MISC_FEATURE

<222>(7)..(18)

<223>Xaa=any one aminoacid

<220>

<221>MISC_FEATURE

<222>(20)..(22)

<223>Xaa=any one aminoacid

<220>

<221>MISC_FEATURE

<222>(23)..(24)

<223>Xaa=any one aminoacid

Xaa can be existed or non-existent

<220>

<221>MISC_FEATURE

<222>(26)..(26)

<223>Xaa=any one aminoacid

<220>

<221>MISC_FEATURE

<222>(27)..(35)

<223>Xaa=any one aminoacid

Xaa can be existed or non-existent

<400>3

Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa His Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa

35

<210>4

<211>5

<212>PRT

<213>Artificial

<220>

<223>ZC fingers

<400>4

Gly Leu Arg Gly Ser

1 5

<210>5

<211>6

<212>PRT

<213>Artificial

<220>

<223>ZC fingers

<400>5

Gly Gly Leu Arg Gly Ser

1 5

<210>6

<211>1199

<212>DNA

<213>Semen Maydiss another name for Sichuan Province (Zea mays)

<400>6

atggagatgg atggggttct gcaagccgcg gatgccaagg actgggttta caagggggaa 60

ggcgccgcga atctcatcct cagctacacc ggctcgtcgc cctccatggt aagcgctgag 120

taggttctta ctgagcgtgc acgcatcgat cacttgactt taggggctca atgtgtgatt 180

cacgggtgcc gcggcgccat tcgagctcca gatccagtac cgctcgagca agtgataaaa 240

catggagcag ggacgatcac gtggtcactt gaaaattacg tgaggtccgg ggcgacgatg 300

tacggcgcgg cgaactctca aacactcaca caaccaaaac cgcttcgtgt tcgtctttgt 360

tccaagcgac tgtgtgagtg tttgagagtt cgccagcgcg acatcgcccg atctgacaaa 420

ttaagctttc gttgcttttc catgattgtg cattttgtga gcatgcactg aatactatga 480

tggatatgtt tggaggaagc attattccaa tttgatgata agggtgttat ttacacttgt 540

tttcagcttg gcaaggtact gcggctcaag aagattctaa aaaacaagtc gcagcgggca 600

ccgagttgta ttgtattctc aagtcatgag caactcctgt ggggccatat cccagaactg 660

gttgagtcgg tcaaacaaga ttgcttggct caagcctatg cagtgcatgt tatgagccaa 720

cacctgggtg ccaatcatgt cgatggtggg gtatggttca gattcagttc atttatgtcc 780

tgttattgtg attttgattg gtaacatatt gacaacctcg acacttggga tcagattcag 840

ttcacttatg gaagaaattg gagaattgtt ataatttatc tataatcacc cctactgaaa 900

tagaaataac atggcatcaa tgtgcatgct attggatttt gacacgaata tgctttattc 960

tatcatatgt tggtaattcc agcaggcagc aggcactact ctttggatcc acgtgacttg1020

acaaagaaat catgccatct ttccacaatg caggtccgtg tacgtgtttc tagggatttt1080

ctggagcttg tcgaaaagaa tgttcttagc agccgtcctg ctgggagagt aaatgcaagt1140

tcaattgata acactgctga tgccgctctt ctaatagcag accactcttt attttctgg 1199

<210>7

<211>50

<212>DNA

<213>Semen Maydiss another name for Sichuan Province

<400>7

caactcctgt ggggccatat cccagaactg gttgagtcgg tcaaacaaga 50

<210>8

<211>12

<212>DNA

<213>Semen Maydiss another name for Sichuan Province

<400>8

ctgtggggcc at 12

<210>9

<211>18

<212>DNA

<213>Semen Maydiss another name for Sichuan Province

<400>9

cttgaccaac tcagccag 18

<210>10

<211>59

<212>DNA

<213>Semen Maydiss another name for Sichuan Province

<400>10

aagtcatgag caactcctgt ggggccatat cccagaactg gttgagtcgg tcaaacaag 59

<210>11

<211>53

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>11

aagtcatgag caactcctgt ggggccaaga actggttgag tcggtcaaac aag 53

<210>12

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>12

His Thr Lys Ile His Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>13

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>13

His Thr Lys Ile Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>14

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>14

His Thr Lys Gly Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>15

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>15

His Thr Lys Ala Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>16

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>16

His Thr Lys Val Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>17

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>17

His Thr Lys Leu Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>18

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>18

His Thr Lys Ser Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>19

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>19

His Thr Lys Asn Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>20

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>20

His Thr Lys Lys Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>21

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>21

His Thr Lys Arg Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>22

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>22

His Thr Lys Ile Gly Gly Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>23

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>23

His Thr Lys Ile Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>24

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>24

His Thr Lys Ile Cys Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>25

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>25

His Thr Lys Ile Gly Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>26

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>26

His Thr Lys Ile Gly Cys Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>27

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>27

His Leu Lys Gly Asn Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>28

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>28

His Leu Lys Gly Asn Cys Pro Ala Gly Ser Gln Leu Val

1 5 10

<210>29

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>29

His Ser Glu Gly Gly Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>30

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>30

His Ser Glu Gly Gly Cys Pro Gly Gly Ser Gln Leu Val

1 5 10

<210>31

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>31

His Ser Ser Ser Asn Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>32

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>32

His Ser Ser Ser Asn Cys Thr Ile Gly Ser Gln Leu Val

1 5 10

<210>33

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>33

His Thr Lys Ile Cys Gly Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>34

<211>16

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>34

His Thr Lys Ile Gly Cys Gly Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>35

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>35

His Thr Lys Ile Gly Gly Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>36

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>36

His Thr Lys Ile Gly Gly Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>37

<211>16

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>37

His Thr Lys Ile Gly Gly Cys Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>38

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>38

His Thr Lys Arg Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>39

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>39

His Thr Lys Arg Cys Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>40

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>40

His Thr Lys Arg Cys Gly Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>41

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>41

His Thr Lys Arg Gly Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>42

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>42

His Thr Lys Arg Gly Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>43

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>43

His Thr Lys Arg Gly Cys Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>44

<211>16

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>44

His Thr Lys Arg Gly Cys Gly Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>45

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>45

His Thr Lys Arg Gly Gly Cys Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>46

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>46

His Thr Lys Arg Gly Gly Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>47

<211>16

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>47

His Thr Lys Arg Gly Gly Cys Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>48

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>48

His Leu Lys Gly Asn Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>49

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>49

His Leu Lys Gly Asn Cys Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>50

<211>16

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>50

His Leu Lys Gly Asn Cys Gly Gly Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>51

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>51

His Lys Glu Arg Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>52

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>52

His Thr Arg Arg Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>53

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>53

His Ala Gln Arg Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>54

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>54

His Lys Lys Phe Tyr Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>55

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>55

His Lys Lys His Tyr Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>56

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>56

His Lys Lys Tyr Thr Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>57

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>57

His Lys Lys Tyr Tyr Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>58

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>58

His Lys Gln Tyr Tyr Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>59

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>59

His Leu Leu Lys Lys Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>60

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>60

His Gln Lys Phe Pro Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>61

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>61

His Gln Lys Lys Leu Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>62

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>62

His Gln Ile Arg Gly Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>63

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>63

His Ile Lys Arg Gln Ser Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>64

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>64

His Ile Arg Arg Tyr Thr Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>65

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>65

His Ile Ser Ser Lys Lys Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>66

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>66

His Lys Ile Gln Lys Ala Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>67

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>67

His Lys Arg Ile Tyr Thr Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>68

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>68

His Leu Lys Gly Gln Asn Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>69

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>69

His Leu Lys Lys Asp Gly Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>70

<211>15

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>70

His Leu Lys Tyr Thr Pro Cys Gly Leu Arg Gly Ser Gln Leu Val

1 5 10 15

<210>71

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>71

His Thr Lys Arg Cys Gly Arg Gly Ser Gln Leu Val

1 5 10

<210>72

<211>14

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>72

His Thr Lys Ile Gly Cys Gly Gly Arg Gly Ser Gln Leu Val

1 5 10

<210>73

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>73

His Leu Lys Gly Asn Cys Gly Arg Gly Ser Gln Leu Val

1 5 10

<210>74

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>74

His Leu Lys Gly Asn Cys Gly Gly Gly Ser Gln Leu Val

1 5 10

<210>75

<211>11

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>75

His Ile Arg Thr Cys Thr Gly Ser Gln Lys Pro

1 5 10

<210>76

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>76

His Ile Arg Thr Cys Gly Thr Gly Ser Gln Lys Pro

1 5 10

<210>77

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>77

His Ile Arg Thr Gly Cys Thr Gly Ser Gln Lys Pro

1 5 10

<210>78

<211>13

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>78

His Ile Arg Thr Gly Cys Gly Thr Gly Ser Gln Lys Pro

1 5 10

<210>79

<211>11

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>79

His Ile Arg Arg Cys Thr Gly Ser Gln Lys Pro

1 5 10

<210>80

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>80

His Ile Arg Arg Gly Cys Thr Gly Ser Gln Lys Pro

1 5 10

<210>81

<211>11

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>81

His Thr Lys Ile His Thr Gly Ser Gln Lys Pro

1 5 10

<210>82

<211>11

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>82

His Thr Lys Ile Cys Thr Gly Ser Gln Lys Pro

1 5 10

<210>83

<211>11

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>83

His Thr Lys Arg Cys Thr Gly Ser Gln Lys Pro

1 5 10

<210>84

<211>11

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>84

His Ala Gln Arg Cys Thr Gly Ser Gln Lys Pro

1 5 10

<210>85

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>85

His Thr Lys Ile Cys Gly Thr Gly Ser Gln Lys Pro

1 5 10

<210>86

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>86

His Thr Lys Arg Cys Gly Thr Gly Ser Gln Lys Pro

1 5 10

<210>87

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>87

His Ala Gln Arg Cys Gly Thr Gly Ser Gln Lys Pro

1 5 10

<210>88

<211>12

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>88

His Thr Lys Ile His Leu Arg Gly Ser Gln Leu Val

1 5 10

<210>89

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>89

His Ala Gln Arg Cys Gly Gly

1 5

<210>90

<211>8

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>90

His Ala Gln Arg Cys Gly Gly Gly

1 5

<210>91

<211>8

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>91

His Thr Lys Ile Cys Gly Gly Gly

1 5

<210>92

<211>8

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>92

His Thr Lys Arg Cys Gly Gly Gly

1 5

<210>93

<211>6

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>93

His Ala Gln Arg Cys Gly

1 5

<210>94

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>94

Cys Cys His Thr Lys Ile His

1 5

<210>95

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>95

Cys Cys His Thr Lys Ile His

1 5

<210>96

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>96

Cys Cys His Thr Lys Ile Cys

1 5

<210>97

<211>10

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>97

Cys Cys His Thr Lys Arg Cys Gly Gly Gly

1 5 10

<210>98

<211>8

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>98

Cys Cys His Ala Gln Arg Cys Gly

1 5

<210>99

<211>8

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>99

Cys Cys His Ile Arg Thr Gly Cys

1 5

<210>100

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>100

Cys Cys His Thr Lys Ile His

1 5

<210>101

<211>8

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>101

Cys Cys His Ile Arg Thr Gly Cys

1 5

<210>102

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>102

Cys Cys His Thr Lys Ile His

1 5

<210>103

<211>9

<212>PRT

<213>Artificial

<220>

<223>Zinc-finger motif

<400>103

Cys Cys His Ala Gln Arg Cys Gly Gly

1 5

<210>104

<211>29

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>104

atggagatgg atggggttct gcaagccgc 29

<210>105

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>105

Asp Arg Ser Ala Leu Ser Arg

1 5

<210>106

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>106

Arg Asn Asp Asp Arg Lys Lys

1 5

<210>107

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>107

Arg Ser Asp Asn Leu Ser Thr

1 5

<210>108

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>108

His Ser His Ala Arg Ile Lys

1 5

<210>109

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>109

Arg Ser Asp Val Leu Ser Glu

1 5

<210>110

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>110

Gln Ser Gly Asn Leu Ala Arg

1 5

<210>111

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>111

Arg Ser Asp Asn Leu Ala Arg

1 5

<210>112

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>112

Thr Ser Gly Ser Leu Thr Arg

1 5

<210>113

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>113

Thr Ser Gly Asn Leu Thr Arg

1 5

<210>114

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>114

Arg Ser Asp His Leu Ser Glu

1 5

<210>115

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>115

Gln Ser Ala Thr Arg Lys Lys

1 5

<210>116

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>116

Glu Arg Gly Thr Leu Ala Arg

1 5

<210>117

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>117

Arg Ser Asp Ala Leu Thr Gln

1 5

<210>118

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>118

Arg Ser Asp Ser Leu Ser Ala

1 5

<210>119

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>119

Arg Ser Ala Ala Leu Ala Arg

1 5

<210>120

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>120

Arg Ser Asp Asn Leu Ser Glu

1 5

<210>121

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>121

Ala Ser Lys Thr Arg Thr Asn

1 5

<210>122

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>122

Asp Arg Ser His Leu Ala Arg

1 5

<210>123

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>123

Arg Ser Asp His Leu Ser Thr

1 5

<210>124

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>124

Gln Ser Gly Ser Leu Thr Arg

1 5

<210>125

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>125

Gln Asn His His Arg Ile Asn

1 5

<210>126

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>126

Thr Gly Ser Asn Leu Thr Arg

1 5

<210>127

<211>7

<212>PRT

<213>Artificial

<220>

<223>Zinc finger calmodulin binding domain CaM

<400>127

Asp Arg Ser Ala Leu Ala Arg

1 5

<210>128

<211>18

<212>DNA

<213>Artificial

<220>

<223>IPP2K zinc finger target sequences

<400>128

gaactggttg agtcggtc 18

<210>129

<211>18

<212>DNA

<213>Artificial

<220>

<223>IPP2K zinc finger target sequences

<400>129

gaactggttg agtcggtc 18

<210>130

<211>12

<212>DNA

<213>Artificial

<220>

<223>IPP2K zinc finger target sequences

<400>130

atggccccac ag 12

<210>131

<211>15

<212>DNA

<213>Artificial

<220>

<223>IPP2K zinc finger target sequences

<400>131

ggcacccagg tgttg 15

<210>132

<211>18

<212>DNA

<213>Artificial

<220>

<223>IPP2K zinc finger target sequences

<400>132

gtcgatggtg gggtatgg 18

<210>133

<211>18

<212>PRT

<213>Artificial

<220>

<223>NLS is derived from Semen Maydiss op-2

<400>133

Arg Lys Arg Lys Glu Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Tyr

1 5 10 15

Arg Lys

<210>134

<211>18

<212>PRT

<213>Artificial

<220>

<223>From the 2A sequences of Thosea asigna viruses

<400>134

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210>135

<211>30

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>135

ggaagcatta ttccaatttg atgataatgg 30

<210>136

<211>29

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>136

cccaagtgtc gaggttgtca atatgttac 29

<210>137

<211>26

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>137

ssscaccaag ttgtattgcc ttctca 26

<210>138

<211>26

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>138

sssataggct tgagccaagc aatctt 26

<210>139

<211>855

<212>DNA

<213>Artificial

<220>

<223>Position -1 3 '-homology flanking sequence

<400>139

gcggccgcta gatagcagat gcagattgct tgcttctctg gtttgatttt tggagtcacc 60

atttctgttt ggttcgtgtg cctcagtgtc tgacagcagc agatcctcga tggagatgga 120

tggggttctg caagccgcgg atgccaagga ctgggtttac aagggggaag gcgccgcgaa 180

tctcatcctc agctacaccg gcacgtcgcc ctccatggta agcgctgagt aggttcttac 240

tgagtgtgca cgcatcgatc acttgacttt aggggctcaa tgtgtgattc acgggtgccg 300

ccattcgagc tccagatcca gtatcgctcg agcaagtgat aaaacatgga gcagggacga 360

tcacgtggtc acttgaaaat tatgtgaggt ccggggcgac gatgtacggc gcggcgaact 420

ctcaaacact cacacagcca aaaccgcttc gtgttcgtct ttgttccaag cgaccgtgtg 480

gtgtgtttgt agtagttcgc cggcgccgca catcgtcgcc ccggatctga caaattaagc 540

tttcgttgct tttccacgat tgtgcatttt ctgagcatgc actgaatact atgatggata 600

tgtttggagg aagcattatt ccaatttgat gataatggtg ttatttacac ttgttttcag 660

cttggcaagg tactgcggct caagaagatt ctaaaaaaca agttgcagcg ggcaccaagt 720

tgtattgcct tctcaagtca tgagcaactc ctgtggggcc atatcccaga actggttgag 780

tcggtcaaac aagattgctt ggctcaagcc tatgcagtgc atgttatgag ccaacacctg 840

ggtgccaata ctagt 855

<210>140

<211>845

<212>DNA

<213>Artificial

<220>

<223>Position -2 3 '-homology flanking sequence

<400>140

actagtcatg tcgatggtgg ggtatggttc agattcagtt catttatgtc ctgttattgt 60

gattttgatt ggtaacatat tgacaacctc gacacttggg atcagattca gttcacttat 120

ggaagaaatt ggagaattgt gataatttat ctataatcac ccctactgaa atagaaataa 180

catgacatca atgtgcatgc tattggattt tgacacgaat atgctttatt ctatcatatg 240

ttggtaattc cagcaggcag caggcactac tctttggatc cacgtgactt gacaaagaaa 300

tcatgccatc tttccacaat gcaggtccgt gtacgtgttt ctagggattt tctggagctt 360

gtcgaaaaga atgttcttag cagccgtcct gctgggagag taaatgcaag ttcaattgat 420

aacactgctg atgccgctct tctaatagca gaccactctt tattttctgg tacgtactct 480

atccctcttc ttaccataat ctgaatcttg ttaaggttta aaatatacga ttgattaagt 540

aaaatccaga gctctattca tatctcacgc actgatgttt tgatgaaacg cttgcagcaa 600

gacggttgcc tgttatttct atttgcatta gacaaacagt cacctttgtt tataaaggtc 660

tttgaatttg cagttcttat aggtttaagt ttgcaactgt tacttacaac agcccaatgg 720

gtagcatcaa gattgttttt ttcagtgatt cataacttaa ctcttggtta aaccgctaga 780

acatggttgg tgtcttaaaa tgcaactggt cctgaggccg taacctgaaa tcattgtacg 840

tcgac 845

<210>141

<211>484

<212>DNA

<213>Artificial

<220>

<223>- IPP2K the gene regions of upstream 5 ' of ZFN targeted areas

<400>141

tggacggagc gagagccaga attcgacgct ggcggcggcg cgtcgccaat acgcagcgcg 60

gatgtggagc cacatgcaaa cgtgtgtccg cccgcgtggc gtccactctc cctccacgtt 120

tcggcgtcct cgtcgccttc ctgggaaatc tccagctact gcccactgcc ccttcccttc 180

agtccctttc cccgggctgt ggtaccagta ctagtaccag catctcttca ggctccacca 240

agcgcagaca ccgcagcagc ggcagcagca cgatccggtg accccccgcc gcgtccagcc 300

tgctcctccg gtgatcgccg gactggcggg gtaggaacca gcggagcgca gcccgcctcc 360

ttccgctggt aagagtgacg cccgcccgct cctcccttcg ctcgcttcct tgctcttccg 420

attctggcgt accagtctca ccgcggcttg gggatttgat gcggagctag ttaaccagca 480

gagc 484

<210>142

<211>729

<212>DNA

<213>Artificial

<220>

<223>- IPP2K the gene orders of downstream 3 ' of ZFN targeted areas

<400>142

attgtttttt tcagtgattc ataacttaac tcttggttaa accgctagaa catggttggt 60

gtcttaaaat gcaactggtc ctgaggccgt aacctgaaat cattgtactt ttctctcatt 120

tctttagata tttccaaaac tctacattag atgatttatg tttgcttact tagtctttct 180

taatctcagg caatcctaag ggtagcagct gcatagctgt agagataaag gtactttgca 240

agcttcctct tttattctta tttttcattt cttatgtata tttctcctca accatttgac 300

ttcttttcgg catgctctac cttgcaggcc aaatgtgggt ttctgccatc atcagaatat 360

atatcagaag ataatactat caagaaacta gtaacgagat ataagatgca tcagcacctc 420

aaattttatc agggtgaggt gtgtagattg gaatgcttga tgccttgatc caagataaaa 480

ttccactctc ttttgcgcac ttaaaaaaca tccatcgatg atacaaactt gatcaaaata 540

ccttaaggct tgttatttac ggcactgttg taatattata ccgtctcttg ctttttgaca 600

tcaggttgat tcccaataca ttcttgcaca catttcagat atcgaagact agtgagtaca 660

atcctcttga tctattttct gggtcaaaag agagaatatg catggccatc aagtcccttt 720

tctcaactc 729

<210>143

<211>42

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>143

gcggccgcgt ctcaccgcgg cttggggatt ggatacggag ct 42

<210>144

<211>38

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>144

actagtgata tggccccaca ggagttgctc atgacttg 38

<210>145

<211>40

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>145

actagtccag aactggttga gtcggtcaaa caagattgct 40

<210>146

<211>32

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>146

gtcgaccttg atgctaccca ttgggctgtt gt 32

<210>147

<211>30

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>147

gcggccgcta gatagcagat gcagattgct 30

<210>148

<211>29

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>148

actagtattg gcacccaggt gttggctca 29

<210>149

<211>38

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>149

actagtcatg tcgatggtgg ggtatggttc agattcag 38

<210>150

<211>37

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>150

gtcgacgtac aatgatttca ggttacggcc tcaggac 37

<210>151

<211>41

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>151

actagttaac tgacctcact cgaggtcatt catatgcttg a 41

<210>152

<211>29

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>152

actagtgtga attcagcact taaagatct 29

<210>153

<211>101

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>153

actagtggcg gcggagaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat 60

cccggcccta ggatggcttc tccggagagg agaccagttg a 101

<210>154

<211>35

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>154

actagtatgc atgtgaattc agcacttaaa gatct 35

<210>155

<211>28

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>155

ctgtggtacc agtactagta ccagcatc 28

<210>156

<211>31

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>156

tcttggatca aggcatcaag cattccaatc t 31

<210>157

<211>20

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>157

tgggtaactg gcctaactgg 20

<210>158

<211>20

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>158

tggaaggcta ggaacgctta 20

<210>159

<211>20

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>159

ccagttaggc cagttaccca 20

<210>160

<211>20

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>160

taagcgttcc tagccttcca 20

<210>161

<211>31

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>161

cttggcaagg tactgcggct caagaagatt c 31

<210>162

<211>26

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>162

atgaagaaag acagggaatg aaggac 26

<210>163

<211>32

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>163

atgaagaaag acagggaatg aaggaccgcc ac 32

<210>164

<211>28

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>164

catggagggc gacgagccgg tgtagctg 28

<210>165

<211>27

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>165

atcgacatga ttggcaccca ggtgttg 27

<210>166

<211>31

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>166

tttcgacaag ctccagaaaa tccctagaaa c 31

<210>167

<211>28

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>167

acaagctcca gaaaatccct agaaacac 28

<210>168

<211>32

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>168

ttcgacaagc tccagaaaat ccctagaaac ac 32

<210>169

<211>29

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>169

tgctaagaac attcttttcg acaagctcc 29

<210>170

<211>32

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>170

gaacattctt ttcgacaagc tccagaaaat cc 32

<210>171

<211>30

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>171

tggacggagc gagagccaga attcgacgct 30

<210>172

<211>31

<212>DNA

<213>Artificial

<220>

<223>Primer

<400>172

gtgcaagaat gtattgggaa tcaacctgat g 31

<210>173

<211>59

<212>DNA

<213>Semen Maydiss another name for Sichuan Province

<400>173

aagtcatgag caactcctgt ggggccatat cccagaactg gttgagtcgg tcaaacaag 59

<210>174

<211>53

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>174

aagtcatgag caactcctgt ggggccaaga actggttgag tcggtcaaac aag 53

<210>175

<211>55

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>175

aagtcatgag caactcctgt ggggccataa gaactggttg agtcggtcaa acaag 55

<210>176

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>176

aagtcatgag caactcctgt ggggccatag aactggttga gtcggtcaaa caag 54

<210>177

<211>56

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>177

aagtcatgag caactcctgt ggggccatac agaactggtt gagtcggtca aacaag 56

<210>178

<211>57

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>178

aagtcatgag caactcctgt ggggccatac cagaactggt tgagtcggtc aaacaag 57

<210>179

<211>52

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>179

aagtcatgag caactcctgt ggggccagaa ctggttgagt cggtcaaaca ag 52

<210>180

<211>57

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>180

aagtcatgag caactcctgt ggggccatat cagaactggt tgagtcggtc aaacaag 57

<210>181

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>181

aagtcatgag caactcctgt ggggccatag aactggttga gtcggtcaaa caag 54

<210>182

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>182

aagtcatgag caactcctgt ggggccatag aactggttga gtcggtcaaa caag 54

<210>183

<211>57

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>183

aagtcatgag caactcct gg tggggccata cagaactggt tgagtcggtc aaacaag 57

<210>184

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>184

aagtcatgag caactcctgt ggggccatag aactggttga gtcggtcaaa caag 54

<210>185

<211>53

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>185

aagcatgagc aactcctgtg gggccataga actggttgag tcggtcaaac aag 53

<210>186

<211>56

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>186

aagtcatgag caactcctgt ggggccatac agaactggtt gagtcggtca aacaag 56

<210>187

<211>57

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>187

aagtcatgag caactcctgt ggggccatat cagaactggt tgagtcggtc aaacaag 57

<210>188

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>188

aagtcatgag caactcctgt ggggccatag aactggttga gtcggtcaaa caag 54

<210>189

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>189

aagtcatgag caactcctgt ggggccacag aactggttga gtcggtcaaa caag 54

<210>190

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>190

aagtcatgag caactcctgt ggggccatag aactggttga gtcggtcaaa caag 54

<210>191

<211>57

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>191

aagtcatgag caactcctgt ggggccatac cagaactggt tgagtcggtc aaacaag 57

<210>192

<211>54

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>192

aagtcatgag caactcctgt ggggccatag aactggttga gtcggtcaaa caag 54

<210>193

<211>55

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>193

aagtcatgag caactcctgt ggggccataa gaactggttg agtcggtcaa acaag 55

<210>194

<211>56

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>194

aagtcatgag caactcctgt ggggccatac agaactggtt gagtcggtca aacaag 56

<210>195

<211>56

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>195

aagtcatgag caactcctgt ggggccatat agaactggtt gagtcggtca aacaag 56

<210>196

<211>56

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>196

aagtcatgag caactcctgt ggggccatac agaactggtt gagtcggtca aacaag 56

<210>197

<211>57

<212>DNA

<213>Artificial

<220>

<223>The Semen Maydiss IPP2K gene orders of mutation are mediated with ZFN

<400>197

aagtcatgag caactcctgt ggggccatac cagaactggt tgagtcggtc aaacaag 57

<210>198

<211>821

<212>DNA

<213>Artificial

<220>

<223>Position -1 5 '-homology flanking sequence

<400>198

gcggccgcgt ctcaccgcgg cttggggatt ggatacggag ctagttaacc agcagagcta 60

gatagcagac gcagatcgct tgcttctctg gtttgatttt tggagtcacc atttctgttt 120

ggttcgtgtg cctcagtgtc tgacagcagc agatcctcga tggagatgga tggggttctg 180

caagccgcgg atgccaagga ctgggtttac aagggggaag gcgccgcgaa tctcatcctc 240

agctacaccg gcacgtcgcc ctccatggta agcgctgagt aggttcttac tgagtgtgca 300

cgcatcgatc acttgacttt aggggctcaa tgtgtgattc acgggtgccg ccattcgagc 360

tccagatcca gtatcgctcg agcaagtgat aaaacatgga gcagggacga tcacgtggtc 420

acttgaaaat tatgtgaggt ccggggcgac gatgtacggc gcggcgaact ctcaaacact 480

cacacagcca aaaccgcttc gtgttcgtct ttgttccaag cgaccgtgtg gtgtgtttgt 540

agtagttcgc cggcgccgca catcgtcgcc ccggatctga caaattaagc tttcgttgct 600

tttccacgat tgtgcatttt ctgagcatgc actgaatact atgatggata tgtttggagg 660

aagcattatt ccaatttgat gataatggtg ttatttacac ttgttttcag cttggcaagg 720

tactgcggct caagaagatt ctaaaaaaca agttgcagcg ggcaccaagt tgtattgcct 780

tctcaagtca tgagcaactc ctgtggggcc atatcactag t 821

<210>199

<211>821

<212>DNA

<213>Artificial

<220>

<223>Position -1 3 '-homology flanking sequence

<400>199

actagtccag aactggttga gtcggtcaaa caagattgct tggctcaagc ctatgcagtg 60

catgttatga gccaacacct gggtgccaat catgtcgatg gtggggtatg gttcagattc 120

agttcattta tgtcctgtta ttgtgatttt gattggtaac atattgacaa cctcgacact 180

tgggatcaga ttcagttcac ttatggaaga aattggagaa ttgtgataat ttatctataa 240

tcacccctac tgaaatagaa ataacatgac atcaatgtgc atgctattgg attttgacac 300

gaatatgctt tattctatca tatgttggta attccagcag gcagcaggca ctactctttg 360

gatccacgtg acttgacaaa gaaatcatgc catctttcca caatgcaggt ccgtgtacgt 420

gtttctaggg attttctgga gcttgtcgaa aagaatgttc ttagcagccg tcctgctggg 480

agagtaaatg caagttcaat tgataacact gctgatgccg ctcttctaat agcagaccac 540

tctttatttt ctggtacgta ctctatccct cttcttacca taatctgaat cttgttaagg 600

tttaaaatat atgattgatt aagtaaaatc cagagctcta ttcatatctc acgcactgat 660

gttttgatga aacgcttgca gcaagacggt tgcctgttat ttctatttgc attagacaaa 720

cagtcacctt tgtttataaa ggtctttgaa tttgcagttc ttataggttt aagtttgcaa 780

ctgttactta caacagccca atgggtagca tcaaggtcga c 821

Claims (19)

1. a kind of zinc finger protein, it includes non-standard zinc finger, wherein the non-standard zinc finger has the spire for involving DNA combinations Divide and wherein at least one zinc finger includes sequence C ys- (X^A)_2-4-Cys-(X^B)₁₂-His-(X^C)_3-5-Cys-(X^D)_l-10 (SEQ ID NO:3), wherein X^AAnd X^BCan be any aminoacid, and wherein be coordinated the C-terminal of residue and including the 3rd zinc in the 3rd zinc The aminoacid sequence of coordination residue is selected from the group：

HIRTCTGSQKP (SEQ ID NO:75) ；

HIRRCTGSQKP (SEQ ID NO:79)；

HTKICTGSQKP (SEQ ID NO:82)；

HAQRCTGSQKP (SEQ ID NO:84) ；

HTKICGTGSQKP(SEQ ID NO:85)；

HAQRCGTGSQKP(SEQ ID NO:87)。

2. a kind of zinc finger protein, it includes multiple zinc fingers, and wherein at least one zinc finger is included according to non-described in claim 1 Specification zinc finger.

3. the zinc finger protein of any one of claim 1 to 2, wherein the zinc finger protein is comprising following any sequences and is transformed into With reference to the target sequence in IPP2-K genes：

DRSALSR (SEQ ID NO:105)；

RNDDRKK (SEQ ID NO:106)；

RSDNLST (SEQ ID NO:107);

HSHARIK (SEQ ID NO:108)；

RSDVLSE (SEQ ID NO:109)；

QSGNLAR (SEQ ID NO:110)；

RSDNLAR (SEQ ID NO:111)；

TSGSLTR (SEQ ID NO:112)；

TSGNLTR (SEQ ID NO:113)；

RSDHLSE (SEQ ID NO:114)；

QSATRKK (SEQ ID NO:115)；

ERGTLAR (SEQ ID NO:116)；

RSDALTQ (SEQ ID NO:117)；

RSDSLSA (SEQ ID NO:118)；

RSAALAR (SEQ ID NO:119)；

RSDNLSE (SEQ ID NO:120)；

ASKTRTN (SEQ ID NO:121)；

DRSHLAR (SEQ ID NO:122)；

RSDHLST (SEQ ID NO:123)；

QSGSLTR (SEQ ID NO:124)；

QNHHRIN (SEQ ID NO:125)；

TGSNLTR (SEQ ID NO:126 )；

DRSALAR (SEQ ID NO:127)。

4. a kind of fusion protein, it includes the zinc finger protein of any one of claims 1 to 3 and one or more functions domain.

5. the fusion protein of claim 4, wherein the functional domain includes cutting half domain, and wherein described fusion protein bag Containing the joint inserted between the cutting half domain and the zinc finger protein.

6. the fusion protein of claim 5, wherein the length of the joint is 5 aminoacid.

7. the fusion protein of claim 6, wherein the aminoacid sequence of the joint is GLRGS (SEQ ID NO:4).

8. the fusion protein of claim 5, wherein the length of the joint is 6 aminoacid.

9. the fusion protein of claim 8, wherein the aminoacid sequence of the joint is GGLRGS (SEQ ID NO:5).

10. a kind of polynucleotide, its coding is according at least one zinc finger protein of any one of claims 1 to 3 or according to right Require at least one fusion protein of 4 to 9 any one.

11. is a kind of for the chromatinic method of targeting incising cell in plant cell, and methods described is included in the cell A pair fusion protein or the multinuclear of the pair of fusion protein of at least one coding according to any one of claim 5 to 9 of expression Thuja acid, wherein：

Within target sequence 10 nucleotide apart of (a) described fusion protein；And

(b) described fusion protein dimerization, and cut the DNA between the target sequence.

A kind of 12. methods of the targeting genetic recombination in host plant cell, methods described includes：

A () expresses a pair according to the fusion protein of any one of claim 5 to 9 or at least one coding in the host cell The polynucleotide of the pair of fusion protein, wherein the target sequence of the fusion protein is present in selected host target genes seat In；And

B () identification shows the recombinant host cell of sequence change in the host target genes seat.

The method of 13. claim 12, methods described further includes exogenous polynucleotide to be imported in the host cell.

The method of 14. claim 13, wherein the exogenous polynucleotide is integrated in the genome of the host plant cell.

The method of 15. any one of claim 12 to 14, wherein it is the mutation being selected from the group that the sequence changes：Hereditary material Deletion, the insertion of hereditary material, the replacement of hereditary material and its any combinations.

The method of 16. any one of claim 11 to 14, wherein the target sequence is in IPP2-K genes.

The method of 17. claim 15, wherein the target sequence is in IPP2-K genes.

18. it is a kind of for reducing plant seed in Phytic Acid Levels method, methods described include by according to claim 16 or 17 method inactivation changes IPP2-K genes.

19. is a kind of for making phosphorus that the method for utilizing can be more metabolized in plant seed, and methods described is included by according to right Require 16 or 17 method inactivation or change IPP2-K genes.