CN105950623B - A kind of double RVD unit modules library efficiently constructed for TALEN and TALEN construction method - Google Patents
A kind of double RVD unit modules library efficiently constructed for TALEN and TALEN construction method Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, are related to a kind of double RVD unit modules library efficiently constructed for TALEN.The present invention is directed to many deficiencies of existing GG-Vector TALEN assemble method, from the beginning devises the TALEN packaging strategy of " double RVD units " (two-RVD unite).The technical scheme is that a kind of double RVD unit modules library efficiently constructed for TALEN, 144 double RVD unit modules, 8 list RVD unit modules and 24 last bit RVD units including independent packaging.The invention discloses a kind of double RVD unit modules libraries based on Golden Gate PCR cloning PCR can construct the TALEN expression vector of targeting 15~19bp any DNA sequence by primary first-order equation.
Description
Technical field
The invention belongs to gene engineering technology fields, are related to a kind of for TALEN (transcriptional activation sample effector nucleic acid
Enzyme) double RVD (repeat make a variation double residues) the unit module library that efficiently constructs and TALEN construction method.
Background technique
Genome editing technique (genome editing) refers to that the particular sequence for genome carries out rite-directed mutagenesis, determines
One technology of the genetic modifications such as point integration, site-directed replacement.Gene sequence is introduced in situ in genome using genome editing technique
The change of column provides convenience research gene function.It is upper in application, animals and plants new product is formulated using genome editing technique
Kind, it can be to avoid uncertain and to protogene group the damage of expression caused by gene radom insertion in existing transgenic technology.
With the appearance of targeted nuclease technology, realizes target gene and precisely orient knockout (knock-out), to obtain target base
Because of the mutant of knockout.Study at present more three technology be ZFN (zinc finger nuclease, Zinc finger nuclease),
TALEN (transcription activator-like effectors nuclease, transcriptional activation sample effector nucleic acid
Enzyme) and CRISPR/cas9 (The clustered, regularly interspaced, short palindromic
Repeats-associated protein systems, the short palindrome repetitive sequence of the regular intervals of cluster and related protein system
System).
TALENs albumen includes two component parts.First component part is from natural TALE (Transcription
Activator-Like Effectors, transcriptional activation sample effector) albumen, N-terminal contains a translocation domain；It is intermediate
It is one section by 1.5~33.5 repetition amino acid sequences that equal TALE unit does not form, each unit is again by 34 amino acid
Composition, wherein 32 amino acid be it is highly conserved, only the 12nd and 13 amino acids can change, can be specifically
In conjunction with a base sequence, therefore the repetition that is otherwise known as of the two amino acid makes a variation double residue (Repeat variant
Diresidue, RVD), 0.5 last unit contains only 20 amino acid of front；The C-terminal of natural TALE albumen contains one
A nuclear localization signal and activating transcription factor can help TALE albumen to enter nucleus from cytoplasm while play transcriptional activation
Effect.By taking the TALE-AvrXs10 albumen of rice leaf spot bacteria secretion as an example, it combines the DNA sequence dna of 19bp in host cell
17.5 units are needed, find that first, its end 5' base is that T does not need TALE protein unit in natural TALE albumen
In conjunction with the last one base is combined by last about 20 amino acid of 0.5 unit.
In December, 2009, the 326th phase of Science have delivered two TALEs albumen energy for cracking plant virus secretion simultaneously
The mechanism of enough specific recognition base sequences, wherein Moscou etc. has obtained TALEs albumen using the method for bioinformatics completely
Identify the rule of base, and Boch etc. has then decoded this " password " with laboratory facilities.They have found in TALEs albumen the 12nd
(asparagine-isoleucine-NI), (Asparagine-Alanine-NA), (histidine-asparate-are combined with 13 amino acids
HD), (Asparagine-Glycine-NG) efficiently can specifically identify base A, G, C, T respectively, and proposing in the future can be as ZFNs
Equally it is transformed into the tool of gene site-directed modification.2010, University of Minnesota Daniel professor Voytas leader's
Laboratory takes the lead in merging the DNA combination relevant domain of TALE with the nuclease domain of FokI, optimizes the connection between the two
Sequence (linker) obtains the targeted nuclease TALEN for having special cleavage activity for specific dna sequence.Hereafter, it studies
Personnel construct using natural TALEs albumen as skeleton for the different biological genes such as people, rat, mouse, zebra fish
DTALEs (design TALE), and reality is combined together with FokI endonuclease, transcription factor and epigenetic modification enzyme
The now pointed decoration to different plant species genome or regulation.
Second component part of TALENs is the IIS type endonuclease FokI from bacterium, when two FokI occur
Activity will be played after dimerization to cut DNA double chain, will start two kinds of repair mechanism processes after DNA Damage, one
Kind is the repair mechanism (NHEJ) of the non-homogeneous dependence in end, and another kind is the repair mechanism (HDR) for relying on homologous recombination.When not having
When the homologous sequence of external source is added, cell just will start NHEJ repair mechanism, and the end DNA that will be switched off is reunited,
But this repair mechanism is a kind of repair process of existing defects, often introduces new mutation, therefore also just reached into
The purpose of row gene knockout.If the homologous base sequence that external source is added at this time can start second of repair mechanism HDR and repair
It is multiple, using homologous repair mechanism, us can be helped to realize that gene is driven in and gene repair, but if the homologous sequence of external source
It is existing defects, then equally can achieve the effect of gene knockout.
Genome directed modification is carried out using TALEN technology to generally comprise 1) according to target gene searching TALEN candidate target
Site；2) it designs for target site sequence and constructs TALE carrier；3) TALEN carrier is obtained with suitable FokI sequence assembling；
4) by complete TALEN vector introduction aim cell；5) clone of directed modification is screened.Shearing in order to guarantee TALEN is special
Property, undershooting-effect is avoided, the target sequence length of TALEN effect is usually chosen in 15~20bp or so.Thus, it is desirable to which TALE is needed
To contain at least 15 duplicate RVD units, the length of nucleotides that encode this TALE will be greater than 1.5kb, and sequence
Repeatability is very strong.Therefore in practical applications, building is assembled by repeating RVD unit, identifies the TALE of specific dna sequence
Carrier just becomes the committed step and difficult point of the technology.
Currently, the TALEN assemble method developed mainly has：The Golden Gate PCR cloning PCR (GG-PCR) of based on PCR,
Golden Gate PCR cloning PCR (GG-Vector) based on traditional plasmid vector, the LIC method based on long cohesive end are based on digestion
The continuous PCR cloning PCR of connection, high-throughput method based on synthesis in solid state etc..Wherein, the reports such as Cermak based on single RVD plasmid
The Golden Gate PCR cloning PCR (GG-Vector) of vector library is the most widely used skill in TALEN design and component composition
Art system (Cermak T, Starker CG, Voytas DF.2015.Efficient design and assembly of
custom TALENs using the Golden Gate platform.Methods Molecular Biology,1239:
133-159.Cermak T,Dolye EL,Christian M,et al.,2011.Efficient design and
assembly of custom TALEN and other TAL effector-based constructs for DNA
targeting.Nucleic Acids Res,39:e82).This method according to RVD identify nucleotide rule (NI → A, HD →
C, NN → G, NG → T, NK → G) and its all possible position in TALE albumen, it constructs respectively mono- comprising 50 RVD
The plasmid vector of element module, 5 last position RVD module plasmid vectors, 13 TALEN assembling intermediate vectors and 4 TALEN tables
Up to the library of skeleton plasmid carrier.The principle of the library GG-Vector assembling TALEN carrier is the target sequence nucleosides according to design
Acid composition, chooses required different RVD unit carriers from library, is generated by these carriers of specific II type endonuclease digestion
A variety of one-to-one, special matched cohesive ends pair, it is primary to connect 8-10 RVD unit simultaneously.
In practical applications, due to being limited by Golden Gate reaction efficiency, RVD in single TALEN group reaction cartridge
The maximum repetition number of unit no more than 10, therefore in order to assemble the TALEN carrier of identification 15-20bp target sequence, just
Have to assemble TALE sequence of two length less than 10 RVD repetitive units respectively, then they are passed through into Golden Gate method
It connects.Complete TALEN assembling flow path has to be split as 2 independent stages, and time-consuming is on or so 5 working days.
This undoubtedly increases experiment consumption, extends construction schedule, also significantly limits the high throughput applications of TALEN technology.
Summary of the invention
The present invention is directed to many deficiencies of existing GG-Vector TALEN assemble method, from the beginning devises " double RVD units "
The TALEN packaging strategy of (two-RVD unite).
The technical scheme is that a kind of double RVD unit modules library efficiently constructed for TALEN, including independently
The double RVD unit modules of 144 of packaging, 8 list RVD unit modules and 24 last bit RVD units；
Adjacent 2 base of described 144 double RVD unit modules any combination for identification；8 lists
RVD unit module is divided into M08 and two groups of M09, every group of 4 list RVD unit modules, and M08 identifies the when for target sequence being 16bp
15 bases, M09 group identify the 17th base for target sequence when 18bp；
24 last bit RVD units are divided into left group and right group, and every group 12, the last bit RVD unit of left group
The monomer I of 3 ' end fusion I heterodimers of Fok, the list of the end of right group last bit RVD unit module 3 ' fusion I heterodimer of Fok
Body II；Left group and right group respectively include 4 last bit RVD units of the 15th bit base of target sequence of identification 15bp length, know
4 last bit RVD units of the target sequence of other 16bp and 17bp length the 16th and the 17th bit base identify 18bp and 19bp length
Target sequence the 18th and 19 bit bases 4 last bit RVD units.
Further, the both ends of double RVD unit modules, list RVD unit module are according to identification base in target sequence
In site sequence end to end cohesive end is set.
Further, double RVD unit modules and list RVD unit module are placed on A/T cloning vector.
Further, the last bit RVD building unit is into carrier for expression of eukaryon, from 5 ' to 3 ' direction of Expression element according to
Secondary includes CaMV35S promoter, the end nuclear localization signal NLS and 5 ' TALE, ccdB toxin gene, last bit RVD unit and 3 ' ends
The monomer I or monomer II of I heterodimer of TALE and Fok.
Specifically, 144 double RVD unit modules have the nucleotides sequence as described in No.1~144 SEQ ID
Column.
Specifically, 8 list RVD unit modules have the nucleotides sequence as described in No.143~152 SEQ ID
Column.
Specifically, 4 last bit RVD units of the 15th bit base of target sequence of the identification 15bp length have such as SEQ
Nucleotide sequence shown in No.153~156 ID；Identify the target sequence the 16th and the 17th bit base of 16bp and 17bp length
4 last bit RVD units have the nucleotide sequence as shown in No.157~160 SEQ ID；Identify the target of 18bp and 19bp length
4 last bit RVD units of sequence the 18th and 19 bit bases have the nucleotide sequence as shown in No.161~164 SEQ ID.
The present invention also provides the methods using module library building TALEN, include the following steps：To one section of target sequence
Column building is directed to the expression vector of 2 target sequences, and the last bit RVD unit of 1 expression vector uses left group, and another 1 expression carries
The last bit RVD unit of body uses right group；Identification 15,17, the expression vector of 19bp length target sequence it is suitable according to base in sequence
Sequence chooses 7,8,9 double RVD unit modules, and identification the 15th, the 17th, the last bit RVD unit module of the 19th bit base；Know
Other 16, the expression vector of 18bp length target sequence chooses 7,8 double RVD unit modules according to base sequence in sequence, identification the
15, single RVD unit module of the 17th bit base, and the last bit RVD unit module of identification the 16th, the 18th bit base；Then
Pass through Golden Gate PCR cloning PCR one-step synthesis.
By the way that two adjacent single RVD unit modules are connected, forming one can recognize that double RVD of two nucleotide are mono-
Element module, while design can be according to the end to end cohesive end of site sequence, finally by it at the both ends of double RVD unit modules
Being building up on suitable plasmid vector and forming one includes 144 double RVD unit module libraries (Fig. 1).Double RVD unit module roots
It is designed according to the regular and all possible position of RVD identification nucleotide, as the bis- RVD unit modules of NI-NI can recognize AA
Nucleotide, when AA base is located at target sequence the one or two, corresponding double RVD unit module carriers are D01-AA：NI-NI
(D01-01-AA)；When AA base is located at target sequence the three or four, corresponding RVD module carrier is D02-AA：NI-NI
(D02-01-AA)；And so on, what all possible target sequence can be easy find in 144 double RVD unit libraries pair
The module carrier answered.
Other than 144 double RVD unit modules, which further comprises 8 list RVD unit modules (Fig. 2).These lists
Only one RVD repetitive unit of RVD unit module is used for penultimate RVD when target sequence is even length and constructs.Such as building
The TALEN carrier of one identification 16bp target sequence, preceding 1~14bp is according to nucleotide combination of two in 144 double RVD unit libraries
In select suitable 7 carriers, the 15th RVD just uses one in single RVD unit module M08, then passes through Golden
Gate reaction connects this 7 double RVD unit modules, 1 list RVD unit module with corresponding last bit RVD expression vector,
It is completed the TALEN expression vector of 16 RVD of needs.
It is assembled into TALEN expression vector for the ease of double RVD unit modules, which also uses 24 left sides and the right side
Side last bit RVD expression vector (Fig. 3).These last bits RVD expression vector uses plant constitutive promoter CaMV 35S promoter,
And the expression of Nos terminator controlling gene, transcript regions contain TALE albumen n end and C-terminal sequence, identification target sequence most
The last bit RVD repetitive sequence of latter position nucleotide, the sequence of nuclear localization signal NLS, the ccdB toxin gene convenient for vector selection
Sequence and I sequence of nuclease Fok for shearing DNA.Different skeleton expression vectors is adapted to identification different length target sequence
The assembling of the left and right side TALEN of column, what the TALEN protein expression vector for completing to assemble can be convenient passes through mediated by agriculture bacillus
Or other methods import in host cell.The activity shear structure domain wild type FokI is homodimer structure, in practical application,
In order to reduce non-specific cleavage, it is often used the FokI shear structure domain that two sequences in left and right have differences, convenient in the cell
Heterodimer is formed after the completion of translation.Therefore, 2 monomers of dimer are distinguished into carrier construction in this application, containing every
The expression vector of one monomer is for the same target sequence or different target sequences in target sequence.
In order to realize this double RVD unit module TALEN assembly systems, by artificial synthesized mode, fully synthetic 144
Sequence of modules (the SEQ ID No.1 of a double RVD units：D01-01-AA to SEQ ID No.144：D09-16-TT), and respectively
It is building up on pUC57 carrier (purchased from Jin Sirui company), forms double RVD unit module plasmid vector libraries.Likewise, artificial close
At sequence of modules (the SEQ ID No.143 of 8 monomer RVD units：M08-01-A to SEQ ID No.152：M09-04-T),
Also it is building up on pUC57 carrier respectively, forms list RVD unit module plasmid vector library.In addition, to a left side for the reports such as zhang
Side and right side TALEN eukaryotic expression skeleton carrier pZHY500 (EL), pZHY501 (ER) be transformed (Zhang Y, Zhang F,
Li X,et al.,2013.Transcription activator-like effector nucleases enable
efficient plant genome engineering.Plant Physiology,161:20-27.).By plant composing type
Promoter CaMV 35S promoter (SEQ ID No.165 35S P) and Nos terminator (SEQ ID No.170Nos-T) pass through
The method of Fusion PCR is connected into respectively in above-mentioned two skeleton carrier, for regulating and controlling the end nuclear localization signal NLS and 5 ' TALE
(SEQ ID No.166NLS+5 ' TALE), ccdB (SEQ ID No.167ccdB) and 3 ' end TALE and left side Fok I (Seq
3 ' TALE+Fok I-L of No.168), 3 ' end TALE and right side Fok I (3 ' TALE+Fok I-R of SEQ ID No.169) table
It reaches.Further by 24 last bit RVD repetitive unit sequences (Seq NO EL/R07-01-Last A to Seq NO EL/ of synthesis
R09-04-Last T) it is connected into above-mentioned left and right side transformation carrier respectively by fusion DNA vaccine, obtain as shown in Figure 3 12
Left side last bit RVD-TALEN skeleton expression vector and 12 right side last bit RVD-TALEN skeleton expression vectors.
For TALEN assembling of the target sequence length greater than 19bp, the carrier of double RVD unit modules can also use
Library needs to carry out two step GG reaction only because segment is less than 10 limitations in GG reaction to complete assembling building.But
In practical applications, if the length of unilateral side TALEN is 19bp, the length of left and right sides TALEN has just reached 38bp, such target
Sequence length segment is enough to deal with the genome complexity of live species, realizes the specific recognition to particular target site and cuts
It cuts, miss target phenomenon is avoided to generate.
Beneficial effects of the present invention：The invention discloses one kind to be based on Golden Gate PCR cloning PCR, passes through primary first-order equation structure
The RVD unit module library of TALEN expression vector is built, which can target 15~19bp any DNA sequence.Based on this pair of
The method of RVD unit module library construction TALEN expression vector effectively simplifies construction step, reduces experiment consumption, mentions
High packaging efficiency, foreshortens to 1 working day for building time-consuming.It can be greatly optimized TALEN group using module library of the invention
It fills a process and reduces cost, convenient and efficient, cost is less, guarantee is provided for the further genralrlization application of TALEN technology, and
It provides the foundation skeleton carrier for the high throughput applications of TALEN technology.
Detailed description of the invention
The permutation matrix of the underlying carrier (16 × 9=144) of Fig. 1 binary RVD module.144 binary RVD modules are with 9 column
The mode of 16 rows is distributed, and is often classified as the position where double RVD, two adjacent nucleotides corresponding to every behavior one double RVD
Sequence.Column represent position of 2 bases of double RVD units combinations in target sequence in figure, such as D01 indicates double RVD units
In conjunction with 2 bases be located at the 1st, 2 of target sequence, 2 bases that D02 indicates that double RVD units combine are located at target sequence
3rd, 4, and so on, D09 indicates that 2 bases that double RVD units combine are located at the 17th, 18 of target sequence.Row in figure
Represent 2 base sequences and the amino acid for identifying 2 bases that double RVD units combine, such as AA：AA indicates double in NI-NI
2 base sequences that RVD unit combines are AA, and NI-NI indicates to combine the amino acid of AA.As the carrier of first row the first row is
D01-AA, when indicating that the one or two bit base positioned at the TALEN is AA, double RVD are NI-NI, and corresponding sequence is SEQ ID
No.1：D01-AA；The carrier of the 4th column the third line is D04-AG, when indicating that the seven or eight bit base positioned at the TALEN is AG,
Double RVD are NI-NN, and corresponding sequence is SEQ ID No.51：D04-AG.CATG,GGAC,CCAG,TGTT,TGCA,CGGT,
GAAA,TCGA,GCTC：The end to end cohesive end in double RVD unit modules both ends；NI-NI,NH-HD,NI-NN,NI-NG,
HD-NI,HD-HD,HD-NN,HD-NG,NN-NI,NN-HD,NN-NN,NN-NG,NG-NI,NG-HD,NG-NN,NG-NG：Identification
Double RVD corresponding DNA sequences of continuous two different nucleotide；Kan：Kalamycin resistance gene.
The underlying carrier (4 × 2=8) of Fig. 2 monomer RVD module.8 list RVD modules are arranged with M08 column and M09 column two, ACGT
The mode that each a line amounts to four rows is distributed.M08 arrange for when target sequence length be 16 when, penultimate (the 15th) base
Corresponding RVD selection；M09 is arranged for when target sequence length is 18, the corresponding RVD of penultimate (the 17th) base to be selected.
Such as construct the long 16bp of target sequence, and the 15th when being the TALEN carrier of G, the monomer RVD module selected is M08-G, corresponding sequence
It is classified as SEQ ID No.147：M08-G.GAAA,TCGA,GCTC：The end to end cohesive end in single RVD unit module both ends；
NI,HD,NN,NG：Identify the RVD corresponding DNA sequence of different mononucleotides；Kan：Kalamycin resistance gene.
The TALEN skeleton expression vector (4 × 3 × 2=24) of Fig. 3 last bit RVD.24 skeleton expression vectors are divided into left side
(EL) it is arranged with right side (ER) TALEN two, is respectively used to use when building left side TALEN carrier and right side TALEN carrier.Each column
(side) carrier identifies that the difference of DNA base is divided into 12 column according to different length target sequence last bit RVD again, and EL/ER07 is used for target sequence
When column length is 15bp, when EL/ER08 for target sequence length is 16bp and 17bp, EL/ER09 is for target sequence length
When 18bp and 19bp.The long 16bp of target sequence is such as constructed, when last bit is carrier on the left of the TALEN of G, the skeleton expression vector selected is i.e.
For EL08-G, corresponding sequence is SEQ ID No.159：EL/R08-Last G.35S-P：Plant constitutive promoter CaMV 35S
Promoter；NLS+5'TALE：Nuclear localization signal+TALE albumen n end sequence；CTAT,GAAA：Cohesive end sequence；ccdB：It is convenient for
The ccdB toxin gene sequence of vector selection；Last-NI：Last bit RVD sequence of modules；3'TALE+FokI-L/R：TALE PROTEIN C
Terminal sequence+for shearing the I left/right sequence of nuclease Fok of DNA；Nos-T：Nos terminator；Amp：Ampicillin is anti-
Property gene.
The building detection of the bis- RVD units of Fig. 4 underlying carrier library (two-RVD unite).A：Double RVD units by taking D04 as an example
Module plasmid vector PCR testing result, 1~16 indicates the bis- RVD unit modules of 16 D04, and M is molecular weight marker；B：8 monomers
RVD unit module plasmid vector PCR testing result, 1~4 indicates the mono- RVD module of 4 M08, and 5~6 indicate the mono- RVD mould of 4 M09
Block, M are molecular weight marker；C：The TALEN skeleton expression vector AatII/AgeI double digestion of last bit RVD by taking 4 EL09 as an example
Testing result, 1~4 indicates that 4 EL09 express skeleton carrier, and M is molecular weight marker.
The left side TALEN expression vector schematic diagram of Fig. 5 rice Os DEP1 gene.35S-P：Plant constitutive promoter CaMV
35S promoter；NLS+5'TALE：Nuclear localization signal+TALE albumen n end sequence；OsDEP1-Left RVD Cluster：Rice
The corresponding left side RVD sequence of DEP1 gene orientation editor's sequence；3'TALE+FokI-L：TALE PROTEIN C terminal sequence+for shearing
The I left side sequence of nuclease Fok of DNA；Nos-T：Nos terminator；Amp：Ampicillin resistance gene；Ori：Replication initiation
Son.
The right side TALEN expression vector schematic diagram of Fig. 6 rice Os DEP1 gene.35S-P：Plant constitutive promoter CaMV
35S promoter；NLS+5'TALE：Nuclear localization signal+TALE albumen n end sequence；OsDEP1-Left RVD Cluster：Rice
The corresponding left side RVD sequence of DEP1 gene orientation editor's sequence；3'TALE+FokI-R：TALE PROTEIN C terminal sequence+for shearing
I right flanks of nuclease Fok of DNA；Nos-T：Nos terminator；Amp：Ampicillin resistance gene；Ori：Replication initiation
Son.
The target gene TALEN expression vector that Fig. 7 is based on the double libraries RVD unit (two-RVD unite) strategies construct and
Detection.TAL-L01, TAL-R01 be table 2 in OsDEP1 gene two TALEN expression vector PCR as a result, the 2# of TAL-L01,
4#, 5#, 6# single colonie have amplified the target stripe (19RVD) of 2000bp or so, and 2#, 3#, 4#, 5#, 6# of TAL-R01 is mono-
Bacterium colony has amplified the target stripe (15RVD) of 1700bp or so；TAL-L02, TAL-R02 are OsBADH2 gene in table 2
Two TALEN expression vector PCR are as a result, 2#, 3#, 4#, 5#, 6# single colonie of TAL-L02 has amplified 1900bp's or so
Target stripe (17RVD)；6 single colonies of TAL-R02 have amplified the target stripe (16RVD) of 1800bp or so；Illustrate 4
A TALEN carrier constructs success.
The target gene TALEN expression vector activity that Fig. 8 is based on double libraries RVD unit (two-RVD unite) strategy is commented
Valence.By protoplast transient expression, detects the shear active from 25% to 55% of different TALEN carriers, can be used for planting
The endogenous gene directed modification of object.
Specific embodiment
It will illustrate the present invention by specific embodiment below, but these specific embodiments and be understood not to
Limitation of the present invention is modified to certain details and is still fallen within protection scope of the present invention.
The underlying carrier in 1 pair of RVD unit library of embodiment constructs and detection
By artificial synthesized mode (synthesis of trust money Si Rui Biotechnology Co., Ltd), fully synthetic 144 double RVD
The sequence of modules (SEQ ID No.1D01-AA to SEQ ID No.144D09-TT) of unit, and it is building up to pUC57 carrier respectively
On (being purchased from Jin Sirui company), double RVD unit module plasmid vectors (Fig. 1) are formed.A series of plasmid vectors are passed through into heat shock method
It is directed respectively into escherichia coli DH5a bacterial strain, obtains double RVD unit module libraries, and each by the amplification of the method for bacterium colony PCR
Double unit areas RVD of plasmid, verify the accuracy in the library.Now it is with 16 carriers of D04-AA to D04-TT that D04 is arranged
Example, introduces its verification method.Using D04-AA bacterium solution as template, corresponding oligonucleotide M13F and M13R is upstream and downstream primer,
Establish following PCR system：10×Taqbuffer5μL,dNTP Mixture(10mM)5μL,M13F(SEQ ID No.171)(10
μM)1μL、M13R(SEQ ID No.172)(10μM)1μL、D04-AA1μL、Taq1μL、ddH2O36μL。
PCR reaction condition is：95 DEG C of initial denaturation, 3min；94 DEG C of denaturation, 20s；56 DEG C of annealing, 20s；Extend 72 DEG C, 15s,
33 circulations, extend 72 DEG C, 3min.The PCR system and reaction condition for verifying other 15 carriers are identical with this, only plasmid mould
Plate and upstream and downstream primer difference.Gained PCR result electrophoresis is as shown in Figure 4 A：Swimming lane 1~16 is respectively the bis- RVD units of 16 D04
The target stripe that module, amplification obtain 200bp or so, is consistent with expection, illustrates 16 double RVD unit module plasmids of D04
Vector construction success.
Likewise, artificial synthesized (synthesis of trust money Si Rui Biotechnology Co., Ltd) module of 8 monomer RVD units
Sequence (SEQ ID No.145M08-A to Seq No.152M09-T), is also building up on pUC57 carrier respectively, and it is mono- to form list RVD
Element module plasmid vector library (Fig. 2).This 8 list RVD module plasmid vectors are directed respectively into Escherichia coli by heat shock method
In DH5a bacterial strain, single RVD module library is obtained, and expands single region RVD of each plasmid by the method for bacterium colony PCR, is verified
The accuracy in the library.PCR system and reaction condition are identical as double RVD unit modules, and only (primer is same for bacterium solution template difference
Upper M13F and M13R).Gained PCR result electrophoresis is as shown in Figure 4 B：Swimming lane 1~4 indicates the mono- RVD module of 4 M08, and 5~6 indicate 4
The target stripe that a mono- RVD module of M09, amplification obtain 100bp or so, is consistent with expection, illustrates 8 lists of M08 and M09
The success of RVD module plamid vector construction.
In order to construct the TALEN skeleton expression vector of 24 last bit RVD, to the left and right side TALEN of the reports such as Zhang
Eukaryotic expression skeleton carrier pZHY500 (EL), pZHY501 (ER) are transformed.Plant constitutive promoter CaMV 35S is opened
Mover (SEQ ID No.16535S P) and Nos terminator (SEQ ID No.170Nos-T) are distinguished by the method for fusion DNA vaccine
It is connected into above-mentioned two skeleton carrier, for regulating and controlling the end nuclear localization signal NLS and 5 ' TALE (SEQ ID No.166NLS+5 '
TALE), the end ccdB (SEQ ID No.167ccdB) and 3 ' TALE and left side Fok I (3 ' TALE+Fok of SEQ ID No.168
I-L), the expression of 3 ' end TALE and right side Fok I (3 ' TALE+Fok I-R of SEQ ID No.169).Further by the 12 of synthesis
A last bit RVD repetitive unit sequence (the last bit RVD of synthesis is 12, in corresponding left side skeleton and right side skeleton carrier this
Partial sequence is FokI difference that is completely the same, only connecting) (SEQ ID No.153EL/R07-Last A to SEQ ID
No.164EL/R09-Last T) it is connected into above-mentioned left and right side transformation carrier respectively by fusion DNA vaccine, it obtains as shown in Figure 3
12 left side last bit RVD-TALEN skeleton expression vectors and 12 right side last bit RVD-TALEN skeleton expression vectors.Fusion
The reaction system of PCR is as follows：10 × KODbuffer5 μ L, dNTP Mixture (10mM), 5 μ L, Primer F (SEQ ID
No.173, SEQ ID No.174 or SEQ ID No.175) (10 μM) 1 μ L, Primer R (SEQ ID No.176) (10 μM) 1 μ
L, pZHY500 (EL)/pZHY501 (ER) 1 μ L, KOD1 μ L, ddH2O36μL.PCR reaction condition is：95 DEG C of initial denaturation, 3min；
94 DEG C of denaturation, 20s；56 DEG C of annealing, 20s；Extend 68 DEG C, 15s, 33 circulations, extends 68 DEG C, 3min.
Whether the skeleton expression vector for verifying building is correct, (is Thermo by AatII/AgeI
The fast enzyme of Scientific.Fermentas) double digestion verified.By taking 4 carriers of EL09 as an example, it is as follows to establish digestion system：
10 × Fast Digest Buffer5 μ L, AatII1 μ L, AgeI1 μ L, plasmid (plasmid) DNA 20 μ L, ddH2O 23μL。
After 37 DEG C of digestion 30min, electrophoresis result is as shown in Figure 4 C, and it is left that 4 EL09 skeleton expression vectors cut out a treaty 4000bp
Right TALEN expression cassette purpose band is consistent with expection, illustrates 24 last bit RVD skeleton expression vector establishments success of EL and ER.
Above-mentioned PCR reaction agents useful for same is that (Shanghai) Biotechnology Co., Ltd KOD-Plus-Neo reagent spins in Japan
Box.
Method and rule of the embodiment 2 based on double RVD unit modules assembling TALEN
When using double RVD unit module tissue T ALEN, target sequence (length is generally in 15~20bp) first is selected, is then set
Corresponding RVD is counted, then is selected suitably from double RVD unit modules library, the list module library RVD and last bit RVD skeleton expression vector
Carrier library, according to Golden Gate reaction constructed.
Double RVD unit modules identify that the regular and all possible position of nucleotide is designed according to RVD, such as NI-
The bis- RVD units of NI can recognize AA nucleotide, and when AA base is located at target sequence the one or two, corresponding RVD module carrier is
For D01-AA：NI-NI(D01-01-AA)；When AA base is located at target sequence the three or four, corresponding RVD module carrier is
For D02-AA：NI-NI(D02-01-AA)；And so on, what all possible target sequence can be easy is mono- in 144 double RVD
Corresponding module carrier is found in first library.And list RVD unit module only one RVD repetitive unit, it is even for target sequence
Penultimate RVD is constructed when number length.The TALEN carrier of an identification 16bp target sequence is such as constructed, preceding 1~14bp is according to core
Thuja acid combination of two selects suitable 7 carriers in 144 double RVD unit libraries, and the 15th RVD just uses monomer RVD
One in unit module M08, then by GG reaction by this 7 binary carriers, 1 monomeric carrier and corresponding last bit RVD table
It is connected up to carrier, is completed the TALEN expression vector of 16 RVD of needs.
Table 1 lists the corresponding RVD module selection rule of different length target sequence, such as assembles a pair and identifies 15bp target sequence
The TALEN of column needs to select the phase that D01 to D07 is arranged in double RVD unit modules library according to DNA sequence dna base composition difference
Plasmid vector is answered, since skeleton expression vector has contained last bit RVD, therefore does not need to select carrier in single RVD unit module, is selected
With corresponding left and right side last bit skeleton expression vector, so that it may assemble the TALEN of complete 15 RVD of left and right side
Carrier.
Table 1 assembles rule based on the TALEN of double RVD unit modules
If the TALEN of same a pair of of identification 16bp target sequence of assembling, different according to DNA sequence dna base composition, in addition to choosing
It selects outside the corresponding plasmid vector that D01 to D07 is arranged in double RVD unit module libraries, it is also necessary to according to the 15th nucleotide in single RVD
A corresponding M08 carrier is selected in unit module, in addition corresponding left and right side last bit RVD skeleton expression vector, so that it may
To assemble the TALEN carrier of 16 RVD of complete left and right side.
TALEN assembling rule for 17~19bp of target sequence length with front 15bp, 16bp be it is similar, it is only double
The plasmid quantity of RVD unit module increases one by one.
For TALEN assembling of the target sequence length greater than 19bp, the carrier of double RVD unit modules can also use
Library needs to carry out two step GG reaction only because segment is less than 10 limitations in GG reaction to complete assembling building.But
In practical applications, if the length of unilateral side TALEN is 19bp, the length of left and right sides TALEN has just reached 38bp, such target
Sequence length segment is enough to deal with the genome complexity of live species, realizes the specific recognition to particular target site and cuts
It cuts, miss target phenomenon is avoided to generate.
The assembling and activity inspection of plant endogenous genes directed modification TALEN carrier of the embodiment 3 based on double RVD unit modules
It surveys
1, the design and assembling of plant endogenous genes directed modification TALEN carrier
To examine the efficiency that using double RVD unit modules plant endogenous genes are oriented with shearing and mutation, selection
OsDEP1 (the GenBank NO. of rice varieties OryzasativaLcv.Nipponbare:FJ039904),OsBADH2(GenBank NO.:KT993490),
OsCKX2(GenBank NO.:AB205193) TaMLO (the GenBank NO. of gene and wheat:KF009556) gene conduct
Target gene has separately designed 4 couples of TALEN and has carried out specific cleavage to its corresponding DNA target sequence.Target gene title, target position
Point DNA sequence dna (No.177~184 SEQ ID), corresponding TALEN title, RVD number, RVD sequence, and in double RVD units
Corresponding binary, monomer and the last bit RVD plasmid vector number that should be selected in library are listed in table 2.
Table 2 constructs situation based on the target gene TALEN expression vector of double RVD unit module packaging strategies
2, the TALEN vector construction and Activity determination of endogenous targets gene
With TAL-L02, TAL- of the TAL-L01 (Fig. 5) of OsDEP1 gene, TAL-R01 (Fig. 6) and OsBADH2 gene
For the building of tetra- TALEN carriers of R02.Based on above-mentioned TALEN design and packaging strategy, with reference to the method for Cermak etc.
(Cermak T,Dolye EL,Christian M,et al.,2011.Efficient design and assembly of
custom TALEN and other TAL effector-based constructs for DNA
targeting.Nucleic Acids Res,39:E82), reacted different RVD and last bit RVD by Golden Gate and
Skeleton expression vector connects.Connection product imports in escherichia coli DH5a through heat shock method, paves plate and is incubated overnight.It chooses respectively
6 monoclonals are selected to carry out bacterium colony PCR detection, as a result as shown in Figure 5：2#, 4#, 5#, 6# single colonie of TAL-L01 expression vector are equal
The target stripe (19RVD) of 2000bp or so is amplified；2#, 3#, 4#, 5#, 6# single colonie of TAL-R01 expression vector expand
Increase the target stripe (15RVD) for 1700bp or so；2#, 3#, 4#, 5#, 6# single colonie of TAL-L02 expression vector expand
The target stripe (17RVD) of 1900bp or so is gone out；6 single colonies of TAL-R02 expression vector have amplified the left side 1800bp
Right target stripe (16RVD).The above result shows that 4 TALEN expression vectors construct success.
The TALEN construction method of other two gene OsCKX2, TaMLO are identical with this, and also pass through bacterium colony PCR inspection
It surveys, it was demonstrated that corresponding TAL-L03, TAL-R03 and TAL-L04, TAL-R04 construct success.
In order to verify the TALEN carrier shear active of building, by every 2 carriers (it is left group that one, which is last bit RVD, one
A last bit RVD is right group) it is used as in one group of importing protoplasm somatocyte, after 2 days dark culturings, the wave under fluorescence microscope
The GFP fluorescence that cell is observed after the blue light excitation of long 450~490nm, counts fluorecyte ratio, obtains above-mentioned 8 TALEN and carries
The specific cleavage Activity Results of body are as shown in Figure 6.8 TALEN carriers show TALEN shear active, activity level
From 25% to 55% etc., show that above-mentioned TALEN carrier may be incorporated in the plant directed modification research of next step.
Claims (8)
1. a kind of double RVD unit modules library for TALEN building, it is characterised in that：144 including independently packing double
RVD unit module, 8 list RVD unit modules and 24 last bit RVD units；
Adjacent 2 base of described 144 double RVD unit modules any combination for identification；8 list RVD are mono-
Element module is divided into M08 and two groups of M09, every group of 4 list RVD unit modules, and M08 identifies the 15th when 16bp for target sequence
Base, M09 group identify the 17th base for target sequence when 18bp；
24 last bit RVD units are divided into left group and right group, and every group 12, the last bit RVD unit 3 ' of left group is held
Merge the monomer I of I heterodimer of Fok, the monomer of the end of right group last bit RVD unit module 3 ' fusion I heterodimer of Fok
Ⅱ；Left group and right group respectively include 4 last bit RVD units of the 15th bit base of target sequence of identification 15bp length, identification
4 last bit RVD units of the 17th bit base of target sequence of the 16th bit base of target sequence and identification 17bp length of 16bp length, know
4 last bit RVD units of the 19th bit base of target sequence of the 18th bit base of target sequence and identification 19bp length of other 18bp length.
2. double RVD unit modules library for TALEN building as described in claim 1, it is characterised in that：Double RVD
Unit module, list RVD unit module both ends be arranged according to site sequence of the identification base in target sequence it is end to end viscous
Property end.
3. double RVD unit modules library for TALEN building as claimed in claim 1 or 2, it is characterised in that：Described is double
RVD unit module and list RVD unit module are respectively placed on A/T cloning vector.
4. double RVD unit modules library for TALEN building as described in claim 1, it is characterised in that：The last bit
For RVD building unit into carrier for expression of eukaryon, from 5 ' to 3 ' direction of Expression element successively includes CaMV 35S promoter, nuclear location
The end signal NLS and 5 ' TALE, ccdB toxin gene, the monomer I of last bit RVD unit and 3 ' end TALE and I heterodimer of Fok
Or monomer II.
5. double RVD unit modules library for TALEN building as claimed in claim 3, it is characterised in that：Described 144
The nucleotide sequence of double RVD unit modules is as shown in No.1~144 SEQ ID.
6. double RVD unit modules library for TALEN building as claimed in claim 3, it is characterised in that：8 lists
The nucleotide sequence of RVD unit module is as shown in No.145~152 SEQ ID.
7. double RVD unit modules library for TALEN building as described in claim 1, it is characterised in that：The identification
The nucleotide sequence such as institute of SEQ ID No.153~156 of 4 last bit RVD units of the 15th bit base of target sequence of 15bp length
Show；Identify 4 last bit RVD of the 16th bit base of target sequence of 16bp length and the 17th bit base of target sequence of identification 17bp length
The nucleotide sequence of unit is as shown in No.157~160 SEQ ID；Identify the 18th bit base of target sequence and the identification of 18bp length
The nucleotide sequence such as institute of SEQ ID No.161~164 of 4 last bit RVD units of the 19th bit base of target sequence of 19bp length
Show.
8. using the method for the described in any item module library building TALEN of claim 1~7, it is characterised in that：Including walking as follows
Suddenly：The expression vector of 2 target sequences is directed to one section of target sequence building, the last bit RVD unit of 1 expression vector uses left side
The last bit RVD unit of group, another 1 expression vector uses right group；Identify the expression vector root of 15,17 or 19bp length target sequence
7,8 or 9 double RVD unit modules are chosen according to base sequence in sequence, and identify the end of the 15th, the 17th or the 19th bit base
Position RVD unit module；Identify that the expression vector of 16 or 18bp length target sequence chooses 7 or 8 pairs according to base sequence in sequence
Single RVD unit module of RVD unit module, identification the 15th or the 17th bit base, and identification the 16th or the 18th bit base
Last bit RVD unit module；Then pass through Golden Gate PCR cloning PCR one-step synthesis.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102770539A (en) * | 2009-12-10 | 2012-11-07 | 明尼苏达大学董事会 | TAL effector-mediated DNA modification |
| CN102787125A (en) * | 2011-08-05 | 2012-11-21 | 北京大学 | Method for building TALE (transcription activator-like effector) repeated sequences |
| CN103131695A (en) * | 2013-02-07 | 2013-06-05 | 西南大学 | Efficient assembling method of transcription activator-like effectors (TALE) repeating region for editing silkworm genome and framework carrier thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770539A (en) * | 2009-12-10 | 2012-11-07 | 明尼苏达大学董事会 | TAL effector-mediated DNA modification |
| CN102787125A (en) * | 2011-08-05 | 2012-11-21 | 北京大学 | Method for building TALE (transcription activator-like effector) repeated sequences |
| CN103131695A (en) * | 2013-02-07 | 2013-06-05 | 西南大学 | Efficient assembling method of transcription activator-like effectors (TALE) repeating region for editing silkworm genome and framework carrier thereof |
Non-Patent Citations (4)
| Title |
|---|
| A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering;Neville E. Sanjana等;《Nat Protoc.》;20130617;第7卷(第1期);第171-192页 * |
| Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting;Tomas Cermak等;《Nucleic Acids Research》;20110414;第1-11页 * |
| Efficient Design and Assembly of Custom TALENs Using the Golden Gate Platform;Tomas Cermak等;《Chromosomal Mutagenesis》;20141103;第133-159页 * |
| TALEN技术研究进展;严爱芬等;《广东农业科学》;20151231(第23期);第145-150页 * |
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