CN104328138A - Method and kit for directional knockout of target gene in genome target - Google Patents

Abstract

The invention relates to the field of gene engineering, and particularly relates to a method and a kit for directional knockout of a target gene in a genome target. The method includes following steps: constructing pPTD-NLS-Cas9; performing in-vitro expression and purification to obtain a pPTD-NLS-Cas9 fusion protein; performing co-incubation to the pPTD-NLS-Cas9 fusion protein with guide RNA to obtain a pPTD-NLS-Cas9/RNA composition; performing co-incubation to the pPTD-NLS-Cas9/RNA composition with a target cell to enable the pPTD-NLS-Cas9/RNA composition to enter the target cell for performing the directional knockout of the target gene. The pPTD-NLS-Cas9/RNA composition, by means of a transmembrane location mechanism of a protein transduction structural domain and a cell nucleus location structural domain, can achieve an effect of quickly and high-efficiently introducing a Cas9 endonuclease and the guide RNA to the target cell, thereby overcoming a technical problem that the Cas9 endonuclease is difficult to introduce to the target cell in the prior art.

Description

The method that the orientation of Genomic targets goal gene knocks out and test kit

Technical field

The present invention relates to genetically engineered field, the method that the orientation in particular to a kind of Genomic targets goal gene knocks out and test kit.

Background technology

Gene targeting is a kind of laboratory facilities of directed change living organisms genetic information.At present, zinc nuclease (ZFN) technology and activating transcription factor sample effector nuclease (TALEN) technology are widely used in gene directed modification, but these technical operating procedures are complicated, cost is high, technical difficulty is comparatively large, and is subject to the restriction of epigenetic modification, cannot carry out target simultaneously knock out for the multiple sites in same cell, therefore, more efficient, cheap and simple gene targeting is urgently developed.

CRISPR (Regularity interval short palindrome tumor-necrosis factor glycoproteins) is the special repetitive dna sequence family of a class.CRISPR is bacterium and Archimycetes is that reply virus and nucleic acid constantly attack the acquired immunity defense mechanism developing and, and is extensively present in numerous prokaryotic gene.Wherein, Equations of The Second Kind CRISPR/Cas system relies on Cas9 endonuclease target and shears foreign DNA.Therefore, CRISPR/Cas system becomes the method for another genome manipulation gradually.

In this system, crRNA (CRISPR-derived RNA) is combined by base pairing and tracrRNA (trans-activating RNA) and forms guide RNA, guide RNA instructs Cas9 albumen to shear double-stranded DNA at crRNA homing sequence target locating point, by inducing cell self DNA repair mechanism, in cellular genome, carry out gene knockout.

In addition, research shows, multiple guide RNA can instruct Cas9 albumen to realize simultaneously to the instantaneous shearing of multidigit point in the genomes such as mammalian cell, human stem cell, yeast.But, relevant in time in, this system relates to the transfection of multiple plasmid or the very large plasmid of molecular weight, and transfection efficiency is low, particularly for the clone of difficult transfection, stem cell, be in the cell etc. of quiescent stage, transfection efficiency and gene knockout efficiency lower.Visible, how Cas9 endonuclease and guide RNA are imported in target cell the important factor becoming and affect genome targeting modification efficiency fast and efficiently; Therefore, provide a kind of can fast, efficiently by Cas9 endonuclease and guide RNA to import in target cell and the method that the orientation of carrying out cellular genome target goal gene the knocks out technical problem that to be people urgently to be resolved hurrily.

Summary of the invention

The method that the orientation that the object of the invention is to a kind of Genomic targets goal gene knocks out, to solve above-mentioned technical problem.

The method that the orientation providing a kind of Genomic targets goal gene in an embodiment of the present invention knocks out, comprises the following steps:

1) the recombinant vectors pPTD-NLS comprising nexin transduction domain PTD, nuclear localization domain NLS, is built; And utilize restriction enzyme digestion connection or recombinant methods in vitro to be connected with Cas9DNA fragment by recombinant vectors, obtain connecting product pPTD-NLS-Cas9;

2), by described connection product conversion in intestinal bacteria, and the vivoexpression recombinant plasmid positive colony comprising PTD, NLS and Cas9 is obtained after carrying out plasmid extraction, qualification; By the described vivoexpression recombinant plasmid positive colony abduction delivering in vitro comprising PTD, NLS and Cas9, after the albumen of expression is purified, obtain PTD-NLS-Cas9 fusion rotein;

3), by described PTD-NLS-Cas9 fusion rotein with can hatch altogether with target goal gene the to be knocked out complementation guide RNA that combine, PTD-NLS-Cas9/RNA mixture is obtained;

4), by described PTD-NLS-Cas9/RNA mixture and target cell hatch altogether, make described PTD-NLS-Cas9/RNA mixture enter described target cell, with apoptotic nueleolus, and the orientation of carrying out goal gene knocks out;

The method that the orientation of this Genomic targets goal gene provided by the invention knocks out, achieve the acquisition of PTD-NLS-Cas9 fusion rotein, wherein, PTD is nexin transduction domain: be that a class is in non-receptor-independent mode, non-classical endocytosis mode directly enters the polypeptide of cell through cytolemma, also referred to as cell-penetrating peptide; Can effectively by many kinds of substance, comprise hydrophilic protein, polypeptide, DNA, RNA even particulate matter etc. be transported at short notice in cell, and not by the restriction of cell type.And NLS is nuclear localization domain can realize entrained macromolecular substance and be positioned at nuclear effect.

Further, then PTD-NLS-Cas9 fusion rotein and the guide RNA that can combine with target goal gene to be knocked out complementation are hatched altogether, obtain PTD-NLS-Cas9/RNA mixture; This mixture contains PTD-NLS-Cas9 fusion rotein and guide RNA simultaneously; Under the effect of PTD-NLS (nexin transduction domain and nuclear localization domain), effect Cas9 endonuclease and guide RNA being imported fast and efficiently target cell core can be realized; In addition, guide RNA can combine with goal gene complementation, and then instruct this mixture orientation to combine, and the effect of the directed double-strand target fragments sheared on goal gene is realized by Cas9, finally by the non-homologous end joining function in self DNA repairing effect of target cell, realize the effect that the genomic efficient fixed point target of target cell knocks out.

This method that the embodiment of the present invention provides, by vivoexpression and purifying obtains PTD-NLS-Cas9 fusion rotein, then hatches PTD-NLS-Cas9 fusion rotein and guide RNA altogether, obtains PTD-NLS-Cas9/RNA mixture; What this mixture utilized nexin transduction domain and nuclear localization domain wears film location mechanism, imports effect in target cell fast and efficiently to realize Cas9 endonuclease and guide RNA; Overcome the technical problem that Cas9 endonuclease in prior art is difficult to import target cell.Can well be applied to and carry out in the actually operating that specific target knocks out, for the aspect such as tumour, viral therapy provides the practical advice method of more convenient and efficient for be correlated with oncogene, the viral genome etc. be incorporated on cell chromosome of tumour.

Optionally, in step 1) in, specifically comprise:

The primer of coding PTD and NLS is annealed, obtains double-stranded DNA, and described double-stranded DNA is inserted into sets out in carrier, obtain the recombinant vectors pPTD-NLS comprising PTD, NLS;

2 restriction endonuclease sites are added at 5 ' and 3 ' end of total length Cas9DNA fragment, and after utilizing Cas9DNA fragment described in digestion with restriction enzyme and described recombinant vectors pPTD-NLS, connect with T4DNA ligase enzyme, obtain vivoexpression recombinant plasmid pPTD-NLS-Cas9.

Or, add vitro recombination enzyme recognition site at total length Cas9DNA fragment two ends, utilize vitro recombination system that total length Cas9DNA fragment is inserted recombinant vectors pPTD-NLS, obtain vivoexpression recombinant plasmid pPTD-NLS-Cas9.

Optionally, in step 1) in, the sequence of the described primer of coding PTD and NLS is as shown in SEQ ID No.1 and SEQ ID No.2.

Optionally, described nexin transduction domain PTD can carry the polypeptide that macromolecular substance carries out cell; Described nuclear localization domain NLS can carry the peptide section that macromolecular substance realizes apoptotic nueleolus.

Optionally, in step 2) in, described external evoked expression adopts protokaryon vivoexpression bacterial strain or eucaryon vivoexpression cell.

Optionally, in step 3) in, the preparation method of described guide RNA comprises: be cloned in cloning vector by the expressed sequence of guide RNA, obtain recombinant cloning vector; With described recombinant cloning vector for template, carry out in-vitro transcription, obtain guide RNA.

Optionally, in step 4) in, described target cell source is eukaryotic cell.

Optionally, in step 4) in, a chain of the target fragments of described goal gene has following structure: 5 '-Nx-NGG-3 ', and wherein, N is any one in A, T, G and C, 19≤X≤30.

The test kit that the orientation realizing the Genomic targets goal gene of aforesaid method knocks out.

Optionally, this test kit comprises:

The encoding gene of Cas9 endonuclease; With the encoding gene of the Cas9 endonuclease of nexin transduction domain and nuclear localization domain;

The recombinant expression vector of the encoding gene containing Cas9 endonuclease; The recombinant expression vector of the encoding gene containing the Cas9 endonuclease with nexin transduction domain and nuclear localization domain;

The Cas9 endonuclease with protein transduction and apoptotic nueleolus function through vivoexpression and after purifying;

Recombinant cloning vector containing the DNA fragmentation be connected in sequence by the coding DNA of promotor, two or more restriction enzyme specific recognition site and guide RNA fragment successively.

Accompanying drawing explanation

In order to be illustrated more clearly in the specific embodiment of the invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.

The method flow schematic diagram that the orientation of the Genomic targets goal gene that Fig. 1 provides for the embodiment of the present invention one knocks out;

The vivoexpression purification result of the PTD-NLS-Cas9 fusion rotein that Fig. 2 provides for the embodiment of the present invention two and western-blot result figure;

The external digestion activity the result figure of the PTD-NLS-Cas9/RNA mixture that Fig. 3 provides for the embodiment of the present invention two;

The TAT PTD-SV40NLS-Cas9/RNA mixture that Fig. 4 provides for the embodiment of the present invention two enters 293T cellular immunofluorescence result figure;

The TAT PTD-SV40NLS-Cas9/RNA mixture that Fig. 5 provides for the embodiment of the present invention two knocks out result figure for hEmx1 gene on 293T cell chromosome.

Embodiment

For making the object, technical solutions and advantages of the present invention clearly; clear, complete description is carried out below by technical scheme of the present invention; based on the embodiment in the present invention; other embodiments all that those of ordinary skill in the art obtain under the prerequisite not making creative work, all belong to the scope that the present invention protects.

Embodiment one

Please refer to Fig. 1, the method that the orientation of this Genomic targets goal gene provided by the invention knocks out, comprise the following steps:

Step 101: build the recombinant vectors pPTD-NLS comprising nexin transduction domain PTD, nuclear localization domain NLS; And utilize restriction enzyme digestion connection or recombinant methods in vitro to be connected with Cas9DNA fragment by recombinant vectors, obtain connecting product pPTD-NLS-Cas9;

In a step 101, by the proteins encoded transduction structural domain after the optimizing according to e. coli codon of customization synthesis and the DNA primer of nuclear localization domain, annealing forms DNA double chain and is inserted into and sets out in vivoexpression carrier, and then constructs recombinant expression vector pPTD-NLS.

Recombinant expression vector pPTD-NLS can be connected with Cas9DNA fragment under the effect of T4DNA ligase enzyme, and then obtains connection product; In order to follow-up conversion and vivoexpression used.

Step 102: product conversion will be connected in intestinal bacteria, and obtain the vivoexpression recombinant plasmid positive colony comprising PTD, NLS and Cas9 after carrying out plasmid extraction, qualification; To the vivoexpression recombinant plasmid positive colony abduction delivering in vitro of PTD, NLS and Cas9 be comprised, after the albumen of expression is purified, obtain PTD-NLS-Cas9 fusion rotein;

Concrete, in a step 102, connection product is first transformed in intestinal bacteria the amplification realizing recombinant plasmid, by the intestinal bacteria after transforming are carried out plasmid extraction, after enzyme cuts qualification, the pPTD-NLS-Cas9 positive colony needed for acquisition.

The pPTD-NLS-Cas9 positive colony obtained proceeds to protokaryon vivoexpression bacterial strain or eucaryon vivoexpression cell again to carry out vivoexpression and purifying and then can obtain the PTD-NLS-Cas9 fusion rotein that can be combined with guide RNA, and the step of expression and purification conveniently operates carries out.

Step 103: PTD-NLS-Cas9 fusion rotein and the guide RNA that can combine with target goal gene to be knocked out complementation are hatched altogether, obtains PTD-NLS-Cas9/RNA mixture;

In step 103, the PTD-NLS-Cas9 fusion rotein after purifying and the guide RNA that can combine with target goal gene to be knocked out complementation are hatched altogether, and then obtain PTD-NLS-Cas9/RNA mixture; This mixture possesses the nucleus effect fast, efficiently entering target cell, and contained Cas9 endonuclease possesses the directed effect sheared.

Step 104: PTD-NLS-Cas9/RNA mixture and target cell are hatched altogether, makes PTD-NLS-Cas9/RNA mixture enter target cell, and with apoptotic nueleolus, and the orientation of carrying out goal gene knocks out.

After PTD-NLS-Cas9/RNA mixture and target cell hatch altogether (incubation time and other processing condition set according to actual effect of hatching), the effect that Genomic targets goal gene orientation knocks out can be realized.

The method that the orientation of this Genomic targets goal gene provided by the invention knocks out, achieve the acquisition of PTD-NLS-Cas9 fusion rotein, wherein, PTD is nexin transduction domain; Can effectively by many kinds of substance, comprise hydrophilic protein, polypeptide, DNA, RNA even particulate matter etc. be transported at short notice in cell, and by the restriction of cell type; And NLS is nuclear localization domain can realize entrained macromolecular substance and be positioned at nuclear effect.

PTD-NLS-Cas9 fusion rotein and with after the guide RNA that combines of target goal gene complementation knocked out be hatched altogether, PTD-NLS-Cas9/RNA mixture can be obtained; Because this mixture contains PTD-NLS-Cas9 fusion rotein and guide RNA simultaneously; Under the effect of PTD-NLS (nexin transduction domain and nuclear localization domain), effect Cas9 endonuclease and guide RNA being imported fast and efficiently target cell core can be realized.Guide RNA can combine with goal gene complementation, and then instruct the directed combination of this mixture, and the effect of the directed double-strand target fragments sheared on goal gene is realized by Cas9, finally by the non-homologous end joining function in self DNA repairing effect of target cell, realize the effect that the genomic efficient fixed point target of target cell knocks out.

In brief, this method that the embodiment of the present invention provides, by vivoexpression and purifying obtains PTD-NLS-Cas9 fusion rotein, then hatches PTD-NLS-Cas9 fusion rotein and guide RNA altogether, obtains PTD-NLS-Cas9/RNA mixture; What this mixture utilized nexin transduction domain and nuclear localization domain wears film location mechanism, imports effect in target cell fast and efficiently to realize Cas9 endonuclease and guide RNA; Overcome the technical problem that Cas9 endonuclease in prior art is difficult to import target cell.Can well be applied to and carry out in the actually operating that specific target knocks out, for the aspect such as tumour, viral therapy provides the practical advice method of more convenient and efficient for be correlated with oncogene, the viral genome etc. be incorporated on cell chromosome of tumour.

The method knocked out in order to the orientation of the Genomic targets goal gene making the above embodiment of the present invention is better applied, more effectively be applied in the modification field of goal gene, the present invention also provides embodiment two on the basis of above-described embodiment, embodiment two, to the further restriction of method and the increase that are above-described embodiment, is now described in detail and explains:

Embodiment two

In the present embodiment, the method that the orientation of Genomic targets goal gene knocks out, comprises the following steps:

S1: the primer of proteins encoded transduction structural domain (PTD) and nuclear localization domain (NLS) is annealed, obtain double-stranded DNA, and double-stranded DNA is inserted into sets out in carrier, obtain the recombinant vectors pPTD-NLS comprising PTD, NLS;

Preferably, the sequence of the primer of proteins encoded transduction structural domain PTD and nuclear localization domain NLS is as shown in SEQ ID No.1 and SEQ ID No.2.

In addition, nexin transduction domain is to carry the polypeptide that macromolecular substance carries out cell; Nuclear localization domain is to carry the peptide section that macromolecular substance realizes apoptotic nueleolus.In the present embodiment, preferred nexin transduction domain is TAT nexin transduction domain, and preferred nuclear localization domain is SV40 nuclear localization domain.

It is pointed out that in further technical scheme, except above-mentioned lifted nexin transduction domain and nuclear localization domain, nexin transduction domain can also comprise: the PTD of naturally occurring PTD and synthetic; Concrete, natural PTD contains a large amount of arginine, lysine residue, and containing a height Pregionp and a spirane structure, typically have the TAT structural domain of HIV-1 virus, Antp, hepatitis B virus albumen PreS2 and pure simplexvirus transcription regulatory protein (VP22) etc.; The PTD of synthetic comprises Transportan, MPG, tenuigenin transduction peptide CTP, RGD, iRGD, MPP, ACPP, NGR, AMP, CREKA peptide, CLT1 peptide, CLT2 peptide etc.

Nuclear localization domain can also comprise: the nuclear localization domain in the albumen such as large T antigen, H2B, v-Jun, NIN2, RB, SWI5, Pho4, rpL25, rpL23a, HnRNP and PTHrP of viral SV40.

Further, in the present invention, for the kind of nexin transduction domain and nuclear localization domain, be not limited to above enumerated example, arbitrary small peptide that macromolecular substance enters cell can be carried all can be used as nexin transduction domain as long as meet; And arbitrary peptide Duan Jun that macromolecular substance realizes apoptotic nueleolus function can be carried can be used as nuclear localization domain.

S2: add 2 restriction endonuclease sites at 5 ' and 3 ' end of total length Cas9DNA fragment, and after utilizing digestion with restriction enzyme Cas9DNA fragment and recombinant vectors pET-TAT PTD-SV40NLS, connect with T4DNA ligase enzyme, obtain connecting product (vivoexpression recombinant plasmid pPTD-NLS-Cas9).

Wherein, total length Cas9DNA fragment, according to Cas9 gene complete sequence, after protokaryon is codon optimized, obtains in the mode of salvage, annealing, connection.

S3: product conversion will be connected in intestinal bacteria, and carry out plasmid extraction, enzyme obtains pET-TAT PTD-SV40NLS-Cas9 positive colony after cutting qualification; PET-TAT PTD-SV40NLS-Cas9 positive colony is proceeded in protokaryon vivoexpression bacterial strain, purifying after vivoexpression, obtain TAT PTD-SV40NLS-Cas9 fusion rotein;

Wherein, the acquisition of pET-TAT PTD-SV40NLS-Cas9 positive colony is consistent with step 102, and therefore not to repeat here, just PTD-NLS is specifically defined as TAT-SV40.And the pET-TAT PTD-SV40NLS-Cas9 positive colony obtained is except containing except carrier pET and total length Cas9 all sequences, also has the sequence with 6 histone labels, a proteins encoded transduction structural domain TAT and nuclear localization domain SV40NLS.

The concrete operations of TAT PTD-SV40NLS-Cas9 fusion rotein vivoexpression and purifying are preferably carried out according to following operation:

A) the positive colony pET-TAT PTD-SV40NLS-Cas9 of acquisition is transformed in E. coli expression strains, after 37 DEG C of overnight incubation, forms single bacterium colony;

B) picking single bacterium colony is in a small amount of liquid nutrient medium, 37 DEG C, after 200rpm shakes incubated overnight, then moves in large volume liquid nutrient medium, large scale culturing, 37 DEG C, and 200rpm concussion is cultured to OD ₆₀₀arrive about 0.6;

C) temperature is adjusted to 10-25 DEG C cultivate and add a certain amount of IPTG, 200rpm shakes overnight induction;

D) add a certain amount of cold lysate after collected by centrifugation thalline, and add a certain amount of protease inhibitor cocktail;

E) with the broken somatic cells of cell sonioation method;

F) with high speed freezing centrifuge, 4 DEG C, collect after centrifugal 30min under 48000 × g condition supernatant in;

G) add the Ni-NTA agarose beads (qiagen company) of 50%, under 4 DEG C of conditions, 2-3h is hatched in concussion;

H) Ni-NTA agarose beads collected by upper separator column, and cleans with the lysate of 5-10 times of column volume;

I) use containing the elution of different imidazole concentration, collect elutriant and detect expression situation and the purity thereof of the Cas9 endonuclease zymoprotein containing protein transduction and nuclear localization domain with SDS-PAGE;

In addition, the albumen obtained by purifying uses His-Tag (2A8) Mouse mAb to carry out western-blot further and verifies that whether it is the Cas9 endonuclease zymoprotein with his fusion tag; Result as shown in Figure 2;

In fig. 2, a result shows that constructed pET-TAT PTD-SV40NLS-Cas9 positive colony great expression can obtain the albumen of about 150kD in escherichia coli expression bacterium.B is the western-blot result expressing the TAT-PTD-SV40NLS-Cas9 fusion rotein obtained, and confirms that this albumen is with his fusion tag, therefore is TAT-PTD-SV40NLS-Cas9 endonuclease enzyme fusion proteins.

SEQ ID No.3 shows the aminoacid sequence of the Cas9 endonuclease zymoprotein (TAT-PTD-SV40NLS-Cas9 fusion rotein) containing protein transduction and nuclear localization domain.

S4: the expressed sequence of guide RNA is cloned in cloning vector; Obtain recombinant cloning vector; Take recombinant cloning vector as template, carry out in-vitro transcription, obtain guide RNA;

Concrete, in this step, enumerate the operation of the guide RNA specific design of targeted human source Emx1 gene, structure and in-vitro transcription, comprise following two aspects:

The construction step of the recombinant cloning vector of A, expression cassette containing guide RNA:

1, design with hEmx1 target fragments and skeleton RNA encoding gene by the principle of 5 '-Nx-NGG-3 ', and obtain the hEmx1gRNA full length sequence with restriction enzyme site sticky end according to the mode of salvage, annealing, connection; Wherein, in SEQ ID No.4 shown in the 53rd – 160 nucleotide sequences be coding hEmx1gRNA full length sequence;

2, being inserted into the hEmx1 target fragments of sticky end and the coding gene sequence of skeleton RNA of obtaining is set out in cloning vector with T7 promotor, build the recombinant cloning vector pET-gRNA-hEmx1 of the expression cassette containing guide RNA;

The in-vitro transcription concrete steps of the recombinant cloning vector pET-gRNA-hEmx1 of B, expression cassette containing guide RNA:

1, suitable digestion with restriction enzyme is used by recombinant cloning vector pET-gRNA-hEmx1 to form linearization plasmid;

2, using the linearization plasmid pET-gRNA-hEmx1 formed as in-vitro transcription template, utilize system-T7Kit in-vitro transcription test kit in-vitro transcription goes out guide RNA, called after hEmx1-gRNA (i.e. guide RNA).

S5: TAT-PTD-SV40NLS-Cas9 fusion rotein and the guide RNA that can combine with target goal gene to be knocked out complementation are hatched altogether, obtains TAT-PTD-SV40NLS-Cas9/RNA mixture.

This step achieves the preparation process of TAT-PTD-SV40NLS-Cas9/RNA mixture, concrete, whether have efficiently only enter target cell rapidly to detect it, and whether there is the effect of external digestion activity, it is as follows that the vitro enzyme of the mixture that PTD-NLS-Cas9 fusion rotein (TAT PTD-SV40NLS-Cas9 endonuclease enzyme fusion proteins) and guide RNA are formed by the present embodiment cuts enzyme work confirmatory experiment concrete steps:

(1) hEmx1-gRNA (guide RNA), in-vitro transcription obtained sex change annealing again;

(2), in upstream and downstream design and synthesis pair of primers hEmx1-F and hEmx1-R (primer sequence SEQ ID No.5 and SEQ ID No.6) of hEmx1 target fragments, with 293T cell genomic DNA for template, PCR post reclaim the hEmx1 gene fragment that acquisition one section comprises target fragments;

(3), get 5 1.5ml EP and manage, be labeled as A, B, C, D, E respectively; Each pipe adds a certain amount of corresponding solution respectively according to table 1;

The mixture of table 1 difference composition

(4), 30min is hatched after above-mentioned A, B, C, D and E pipe mixing in applicable temperature water bath;

(5), add the pure water of damping fluid and a certain amount of DEPC process in A pipe, all the other each pipes add a certain amount of hEmx1 gene fragment comprising target fragments; Be applicable to temperature continuation water-bath after mixing and hatch 2h;

(6), by above-mentioned A, B, C, D and E pipe be placed in 95 DEG C of heating 10min, slow cooling is to room temperature;

(7), respectively take a morsel sample from each pipe, adds after appropriate 6 × DNALoading buffer containing GelRed mixing is splined on 5%PAGE, 150V electrophoresis 30min;

(8), electrophoresis is taken pictures under terminating rear gel imaging instrument ultraviolet condition observation.

The external digestion activity the result of TAT PTD-SV40NLS-Cas9 endonuclease enzyme fusion proteins is shown in Fig. 3.Fig. 3 result shows to only have after TAT PTD-SV40NLS-Cas9 (pET-PTD-NLS-Cas9 fusion rotein) and hEmx1-gRNA (guide RNA) form mixture could specific recognition hEmx1 target fragments cut by 684bp hEmx1 and form 367bp and 317bp two small segments.Therefore, confirm that the TAT PTD-SV40NLS-Cas9 endonuclease enzyme fusion proteins that purification obtains has external digestion activity.

S6: TAT-PTD-SV40NLS-Cas9/RNA mixture and target cell are hatched altogether, makes TAT-PTD-SV40NLS-Cas9/RNA mixture enter target cell, and with apoptotic nueleolus, and the orientation of carrying out goal gene knocks out.

In this step, preferably, target cell takes from eukaryotic cell, is specially mammalian cell, and, in the process of concrete operations, preferably, a chain of the target fragments of goal gene has following structure: 5 '-Nx-NGG-3 ', wherein, N is any one in A, T, G and C, 19≤X≤30.

Further, the present invention efficiently enters mammalian cell effect to TAT-PTD-SV40NLS-Cas9/RNA mixture (mixture that TAT PTD-SV40NLS-Cas9 endonuclease zymoprotein and guide RNA are formed) and verifies:

The PTD-NLS-Cas9/RNA mixture that obtained by vivoexpression purifying (be specially TAT PTD-SV40NLS-Cas9 endonuclease zymoprotein in the present embodiment and hEmx1-gRNA forms mixture) imports 293T cell afterwards, mixture enters cell effect to utilize cellular immunofluorescence to verify, concrete steps are as follows:

(1), 293T cell is inoculated in a CELLview glass bottom Tissue Culture Dish (Greiner Bio-one company) with certain cell density, adds perfect medium, 5%CO ₂24h cultivated by incubator 37 DEG C;

(2), get 5 μ l 10X damping fluids, TAT PTD-SV40NLS-Cas9 endonuclease enzyme fusion proteins and in-vitro transcription hEmx1-gRNA and use DEPC-H ₂after O polishing to 50 μ l mixes, applicable temperature hatches certain hour;

(3), get the above-mentioned complex solution of 40 μ l and join in CELLview glass bottom Tissue Culture Dish, 5%CO ₂incubator 37 DEG C continues to cultivate;

(4), fresh perfect medium is changed, 5%CO after 24h ₂incubator 37 DEG C continues to cultivate;

(5), from cell culture incubator, take out CELLview glass bottom Tissue Culture Dish after 24h, discard substratum;

(6), 3 times are washed, each 10min with 1 × PBS of pre-temperature or room temperature;

(7), the cold formaldehyde room temperature fixed cell certain hour of 4%;

(8), 1 × PBS washed cell 3 times, each 10min;

(9), 0.2%Triton X-100 permeable membrane 10-20min;

(10) 1 × PBS washed cells 3 times, each 10min;

(11), 5%FBS 37 DEG C of closed 30min;

(12), 1 × PBS washed cell 3 times, each 10min;

(13), add the HIV-1Tat Antibody (Millipore company) diluted by a certain percentage, hatch certain hour for 37 DEG C;

(14), 1 × PBS washed cell 3 times, each 10min;

(15), add the goat anti-mouse IgG-FITC (CST company) diluted by a certain percentage, hatch certain hour for 37 DEG C;

(16), 1 × PBS washed cell 3 times, each 10min;

(17), fluorescence microscopy Microscopic observation fluorescence intensity;

(18), with after DAPI (sigma company) transfect cell core, ultraviolet visualization cell under fluorescent microscope.

TAT-PTD-SV40NLS-Cas9/RNA mixture enters 293T cellular immunofluorescence result and please refer to Fig. 4.Fig. 4 shows that the TAT-PTD-SV40NLS-Cas9/RNA mixture of external formation can efficiently enter in the 293T cell of nearly 100%, and is positioned on nucleus.

In addition, the embodiment of the present invention is also verified the effect of genomic knockout in mammalian cell TAT-PTD-SV40NLS-Cas9/RNA mixture (mixture that TAT PTD-SV40NLS-Cas9 endonuclease zymoprotein and guide RNA are formed):

The TAT PTD-SV40NLS-Cas9 endonuclease zymoprotein that vivoexpression purifying obtains and hEmx1-gRNA incubated in vitro, after being combined into mixture, join in 293T cell culture fluid, after hatching 72h altogether, knock out result with surveyor assay methods analyst TAT-PTD-SV40NLS-Cas9/RNA complexes upon cell native gene hEmx1, its concrete steps are as follows:

(1), 293T cell is laid on 24 orifice plates with certain cell density, 5%CO ₂incubator 37 DEG C cultivation;

(2) 5 μ l 10X damping fluids, a certain amount of TAT PTD-SV40NLS-Cas9 endonuclease enzyme fusion proteins and a certain amount of in-vitro transcription hEmx1-gRNA, is got, and after mixing with DEPC-H2O polishing to 50 μ l, applicable temperature forms TAT-PTD-SV40NLS-Cas9/RNA mixture after hatching certain hour; (concrete, the mol ratio of TAT PTD-SV40NLS-Cas9 endonuclease enzyme fusion proteins and in-vitro transcription hEmx1-gRNA can be: 1:0.5 ~ 1:5, and the present embodiment is more excellent is 1:2).

(3), get 40 μ lTAT-PTD-SV40NLS-Cas9/RNA mixtures and join as experimental port in one of them 24 hole 293T cell, another 24 hole 293T cell adds the mixture reaction solution hole in contrast of same volume, 5%CO ₂incubator 37 DEG C continues to cultivate;

(4), after 24h, holes is changed fresh complete medium simultaneously and is continued to cultivate 48h;

(5), cell continues to cultivate after 48h, collects experimental port and control wells cell respectively, uses genome extraction agent box (Di Da bio tech ltd, Shanghai), extract genomic dna according to operation steps shown in test kit;

(6), with this genomic dna for template, utilize two Auele Specific Primer hEmx1-F and hEmx1-R (primer sequence SEQ ID No.5 and SEQ ID No.6), obtain one section of 684bp DNA fragmentation by polymerase chain reaction (PCR) amplification;

(7), PCR primer after 1% sepharose is separated, use glue to reclaim test kit (Di Da bio tech ltd, Shanghai) and also reclaim DNA product according to operation steps shown in this test kit;

(8), reclaim the DNA product that obtains again after sex change annealing, use surveyor assay kit (Transgenomic company) to carry out endonuclease reaction according to operation shown in this test kit;

(9) add after appropriate 6 × DNA Loading buffer containing GelRed mixing is splined on 5%PAGE, respectively, 150V electrophoresis 30min;

(10), electrophoresis is taken pictures under terminating rear gel imaging instrument ultraviolet condition observation.

Knocking out of hEmx1 the results are shown in Figure 5.In Fig. 51 is surveyor assay result display DNA product cut formation 367bp and 317bp two small segments of experimental port; And 2 in Fig. 5 is cut formation small segments of surveyor assay result display DNA product of control wells.Therefore show with experimental port cell genomic DNA obtain DNA fragmentation in target sequence through TAT-PTD-SV40NLS-Cas9/RNA cutting after due to lack recovery template, the main mode with non-homogeneous restructuring is repaired, more or less can insert or delete some bases, thus confirm that the TAT PTD-SV40NLS-Cas9 endonuclease zymoprotein that vivoexpression purifying obtains and the mixture that hEmx1-gRNA is formed can knock out by Experimental genomics target of fixing a point in cell.

The method provided to make above-described embodiment is better realized, the test kit that the orientation that the present invention also provides a kind of Genomic targets goal gene according to the method shown in above-described embodiment knocks out, to implement the methods.

Concrete, in this test kit, comprise the encoding gene of Cas9 endonuclease; With the encoding gene of the Cas9 endonuclease of nexin transduction domain and nuclear localization domain;

The recombinant expression vector of the encoding gene containing Cas9 endonuclease; The recombinant expression vector of the encoding gene containing the Cas9 endonuclease with nexin transduction domain and nuclear localization domain;

The Cas9 endonuclease with protein transduction and apoptotic nueleolus function through vivoexpression and after purifying;

Recombinant cloning vector containing the DNA fragmentation be connected in sequence by the coding DNA of promotor, two or more restriction enzyme specific recognition site and skeleton RNA fragment successively; Needed for the operation of the method that the orientation that the sample shown in above-mentioned can meet Genomic targets goal gene knocks out.

Directed knockout technique protein transduction oligopeptides (structural domain) and Cas9 endonuclease are recombinated and vivoexpression by novelty of the Genomic targets goal gene of the embodiment of the present invention, then assemble to be formed with guide RNA and merge Cas9/RNA mixture, import fast and efficiently in target cell under the help of nexin transduction domain, specifically the genome in cell is knocked out.

This test kit provided by the invention compares other commercialization gene knockout test kits, has high-level efficiency, low toxicity, easy and simple to handle, especially for the clone of difficult transfection, stem cell and the cell etc. being in quiescent condition.Utilize this test kit can to tumour be correlated with oncogene, be incorporated into intracellular viral genome and carry out specific target and knock out, for the aspect such as tumour, viral therapy provides the instrument of more convenient and efficient.

The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. the method that knocks out of the orientation of Genomic targets goal gene, is characterized in that, comprise the following steps:

1) the recombinant vectors pPTD-NLS comprising nexin transduction domain PTD, nuclear localization domain NLS, is built; And utilize restriction enzyme digestion connection or recombinant methods in vitro to be connected with Cas9DNA fragment by recombinant vectors, obtain connecting product pPTD-NLS-Cas9;

2), by described connection product conversion in intestinal bacteria, and the vivoexpression recombinant plasmid positive colony comprising PTD, NLS and Cas9 is obtained after carrying out plasmid extraction, qualification; By the described vivoexpression recombinant plasmid positive colony abduction delivering in vitro comprising PTD, NLS and Cas9, after the albumen of expression is purified, obtain PTD-NLS-Cas9 fusion rotein;

3), by described PTD-NLS-Cas9 fusion rotein with can hatch altogether with target goal gene the to be knocked out complementation guide RNA that combine, PTD-NLS-Cas9/RNA mixture is obtained;

4), by described PTD-NLS-Cas9/RNA mixture and target cell hatch altogether, make described PTD-NLS-Cas9/RNA mixture enter described target cell, with apoptotic nueleolus, and the orientation of carrying out goal gene knocks out.

2. method according to claim 1, is characterized in that, in step 1) in, specifically comprise:

The primer of coding PTD and NLS is annealed, obtains double-stranded DNA, and described double-stranded DNA is inserted into sets out in carrier, obtain the recombinant vectors pPTD-NLS comprising PTD, NLS;

2 restriction endonuclease sites are added at 5 ' and 3 ' end of total length Cas9DNA fragment, and after utilizing Cas9DNA fragment described in digestion with restriction enzyme and described recombinant vectors pPTD-NLS, connect with T4DNA ligase enzyme, obtain vivoexpression recombinant plasmid pPTD-NLS-Cas9.

3. method according to claim 2, is characterized in that, in step 1) in, the sequence of the described primer of coding PTD and NLS is as shown in SEQ ID No.1 and SEQ ID No.2.

4. method according to claim 3, is characterized in that, described nexin transduction domain PTD can carry the polypeptide that macromolecular substance carries out cell;

Described nuclear localization domain NLS can carry the peptide section that macromolecular substance realizes apoptotic nueleolus.

5. method according to claim 1, is characterized in that, in step 2) in, described external evoked expression adopts prokaryotic expression bacterial strain or eukaryotic expression cell.

6. method according to claim 1, is characterized in that, in step 3) in, the preparation method of described guide RNA comprises:

The expressed sequence of guide RNA is cloned in cloning vector, obtains recombinant cloning vector; With described recombinant cloning vector for template, carry out in-vitro transcription, obtain guide RNA.

7. method according to claim 1, is characterized in that, in step 4) in, described target cell source is eukaryotic cell.

8. the method according to any one of claim 1-7, is characterized in that, in step 4) in, a chain of the target fragments of described goal gene has following structure:

5 '-Nx-NGG-3 ', wherein, N is any one in A, T, G and C, 19≤X≤30.

9. the test kit that knocks out of the orientation realizing the Genomic targets goal gene of method according to claim 1.

10. test kit according to claim 9, it is characterized in that, this test kit comprises:

The encoding gene of Cas9 endonuclease; With the encoding gene of the Cas9 endonuclease of nexin transduction domain and nuclear localization domain;

The recombinant expression vector of the encoding gene containing Cas9 endonuclease; The recombinant expression vector of the encoding gene containing the Cas9 endonuclease with nexin transduction domain and nuclear localization domain;

The Cas9 endonuclease with protein transduction and apoptotic nueleolus function through vivoexpression and after purifying;

Recombinant cloning vector containing the DNA fragmentation be connected in sequence by the coding DNA of promotor, two or more restriction enzyme specific recognition site and guide RNA fragment successively.