CN105647885B - Cas9 fusion protein and coding sequence thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and relates to a fusion protein and a DNA sequence for encoding the fusion protein. Specifically, the invention provides a Cas9 fusion protein comprising a Cas9 fragment and a Ubiquitin fragment and a coding sequence thereof. On one hand, the invention can ensure that the Cas9 protein is rapidly degraded after playing a targeting role in embryonic cells, thereby reducing the chimeric mutation effect of embryonic development. More importantly, the Ubiquitin-i-Cas9(i is the first amino acid of a linker connecting the Ubiquitin and the Cas9, and can be arginine (R) or proline (P) or leucine (L)) protein provided by the invention can improve the homozygous efficiency of gene targeting embryos. Particularly preferred Ubiquitin-R-Cas9 protein, the targeting efficiency of the wild-type Cas9 protein gene is improved by 3.51 times. This is beyond the expectation of the skilled person.

Description

Cas9 fusion proteins and its coded sequence

Technical field

The invention belongs to biological technical field, it is related to the DNA sequence dna of a kind of fusion protein and encoding fusion protein.

Technical background

Animal disease model serves key effect in research human diseases pathogenesis and drug screening.Inhuman spirit is long Class animal is highly similar to the mankind on biology, science of heredity, and such as macaque is up to 93% (Gibbs with human genome homology Et al., 2007), thus the important animal model as the research of mankind's major disease and drug screening.However, big due to lacking The embryonic stem cell line of animal, traditional gene targeting is difficult to for setting up primate disease model.And in recent years The gene editing new technology CRISPR/Cas9 of development carries out having shown huge potentiality in accurately modification in orientation to gene, Have been widely used for different genera and carry out genome manipulation and genetic modification.Meanwhile, the new technique is also attempted for treating Some disease (Schwank et al., 2013 caused by gene defect；Wu et al.,2013).But, CRISPR/Cas9 Technology still awaits improving on the precision of genetic modification.

CRISPR/Cas9 is a kind of by RNA to instruct Cas albumen to enter edlin to gene from bacterium acquired immunity With the new technology of modification.Jinek etc. carries out artificial reconstructed allow the system to more simply and easily to CRISPR/Cas9 systems For gene editing (Jinek et al., 2013).Cas9 restriction endonucleases are under the guiding of guide RNA molecule to specific site DNA is cut, and forms double-stranded DNA breach, then cell can be by homologous recombination machinery or non-homologous end joining mechanism The DNA of fracture is repaired.If cell is repaired by homologous recombination machinery, it can fill up disconnected with other section of DNA fragment The DNA breach split, thus one section of " new " hereditary information can be introduced.At present, the Cas9 systems of RNA mediations can be mediated successfully carefully Bacterium, plant cell, zebrafish embryo, mouse, human archeocyte, non-human primate machin embryo, the even mankind discard embryo Genome editor (the Cho et al., 2013 of tire etc.；Cong et al.,2013；Jinek et al.,2013；Mali et al.,2013；Niu et al.,2014；Platt et al.,2014；Shan et al.,2013；Chen et al., 2015).And the technology can also successfully correct dcc gene (the Schwank et al., 2013 of diseased n animal by gene knock-in； Wu et al., 2013) or be reorganized as viral vector and set up animal model for cancer (Platt et al., 2014) etc., previous Achievement in research will all provide hope for technology future applied to the treatment of human inheritance's deficiency disorders.

CRISPR/Cas9 systems develop rapidly so that the foundation of animal disease model be no longer limited to minority patterns life Thing so that realize that genome editor is possibly realized in all species.The technology is easy to use, simple and target practice efficiency is higher, It can realize and by a pair of alleles shear obtaining homozygous mutation, can also design a variety of in multiple sites of target gene GRNA knocks out purpose completely to reach.But there is also latent gene mutation chimeric polymorphism, effect of missing the target, gene at present for the technology Knock in the more low weak point of homologous recombination efficiency.For the chimeric effect problem of gene targeting, due to small animal model growth Cycle is shorter, can by embryonic stem cell screening or constantly hybridization pass on and obtain Mutants homozygous, but for the mankind more For close primate, its sexal maturity time is accomplished by 4 to 5 years, and pregnant time is up to 165 days, and is still lacked at present Monkey embryonic stem cell line, therefore chimeric effect has huge challenge setting up on homozygous gene mutation monkey model.

Occurring for chimeric mutational effect may be because the Cas9mRNA and sgRNA of injection be still held after embryonated egg embryo divides Continued reaches, and this chimerism (Platt et al., 2014 also occurs in injection Cas9 albumen；Sung et al.,2014) (Sung YH,2014；Kim S, 2014), show that the half-life period of Cas9 albumen in the cell is longer so as to causing fertilized egg cell Split into four cells, eight cells even blastaea period when, each cell is formed because being randomly assigned different degrees of Cas9 albumen The animal of different degrees of several genes modified types, the embryonic tissue formed and new life can be in different cell or tissues The different genetic modification type of carrying or the genetic modification for having different degrees of (single-stranded or double-stranded DNA).In addition, cell itself DNA damage repair mechanism or the non-homogeneous recombinantal repair of DNA etc. can also influence gene targeting efficiency and chimeric effect.

Every kind of protein in protokaryon and eucaryote body has its life characteristics, also known as half-life period.Protein Half-life period is relevant with the amino acid of its polypeptide chain N- terminal specifics, i.e., N- hold-carryings are then (Bachmair et al., 1986).Therefore can Using N- hold-carryings then and the ubiquitin fusion protein with variety classes amino acid (ubiquitin fusion degradation, UFD) degrade signal to change the protein half life dependent on ubiquitination degradation pathway, such as N-terminal is connected with Arginine (Ub- R-trageted protein), Leucine (Ub-L--trageted protein), Proline (Ub-P--trageted Protein) or Lys (Ub-K--trageted protein) can substantially reduction destination protein stability so as to accelerate drop Solution, and is connected with Met, Ser, and Ala then causes destination protein more to stablize (Johnson et al., 1995).And meet N- end gauages Its degraded of albumen then is lysine dependent on the amino acid of the 15th or 17, therefore can remove stable degraded signal and mesh Albumen between insertion one section of random amino acid sequence cause the lysine of destination protein to appear in the 17th.And gone in connection If the 3rd lysine on linker between stable degraded signal and destination protein can then further enhance ubiquitination effect Rate (Bachmair et al., 1986；Dantuma et al.,2000).

The content of the invention

Present invention aims at provide a kind of Cas9 fusion proteins, its coded sequence, plasmid and table containing coded sequence Up to carrier.

The Cas9 fusion proteins that the present invention is provided include Cas9 fragments and Ubiquitin fragments.Ubiquitin's and Cas9 The amino acids of connexon the 1st difference can obtain different experiment effects.Described Ubiquitin be Ubiquitin-L, Ubiquitin-P or Ubiquitin-R (L, P, R are the abbreviation of the Ubiquitin and Cas9 amino acids of connexon the 1st).

Ubiquitin DNA encoding sequence is connected in Cas9 DNA encoding sequence by way of plasmid is transformed, So as to reach the effect for accelerating Cas9 protein degradations.The Ubiquitin and Cas9 amino acids difference of connexon the 1st can be obtained not With practical function, by test we have found that Ubiquitin-R (R represents Ubiquitin and Cas9 the 1st ammonia of connexon Base acid is arginine) best results, Ubiquitin-L, Ubiquitin-P take second place.

In a preferred embodiment in accordance with this invention, Ubiquitin is Ubiquitin-R.

As preferred embodiment, described fusion protein contains the amino acid sequence as shown in SEQ ID NO.1.Invention is same When provide a kind of Cas9 fusion proteins coded sequence, it is characterised in that contain Cas9DNA fragments and Ubiquitin DNA pieces Section.

By test we have found that Ubiquitin-R (R represents that the Ubiquitin and Cas9 amino acids of connexon the 1st are Arginine) best results, Ubiquitin-L, Ubiquitin-P take second place.

In a preferred embodiment in accordance with this invention, described Ubiquitin is Ubiquitin-R.

As preferred embodiment, described coded sequence contains the DNA sequence dna as shown in SEQ ID NO.2.

Invention is simultaneously there is provided the plasmid comprising above-mentioned coded sequence, expression vector or host cell；Preferably, it is described Carrier be Ubiquitin-R-Cas9.

A kind of gene targeting method, gene targeting is carried out using the fusion protein or the coded sequence.Preferably, institute Gene is stated for mammalian genes；It is highly preferred that the gene is non-human mammal gene；It is further preferred that described Gene is non-human primate's gene.

A kind of to reduce the method that zooblast gene targeting is fitted together to effect, it is entered using above-mentioned fusion protein or coded sequence Row gene targeting；Preferably, described cell is mammalian cell；It is highly preferred that described cell is dynamic for non-human lactation Thing cell；It is further preferred that described cell is non-human primate's cell

As one embodiment of the present invention, CRISPR/Cas9 is significantly reduced in inhuman spirit the invention provides one kind Long class animal machin gene targeting is fitted together to the new method of effect：Shortened by adding ubiquitination degraded signal Ubiquitin The half-life period of Cas9 albumen, it was only played in development of fertilized ova early stage (two cell stages or four cell stages) with quantitative control Gene targeting is acted on.

In a particularly preferred embodiment of the present invention, disclose and a kind of specifically degraded signal containing Ubiquitin The transformation carrier of Cas9 protein sequences and its application in primate machin.Specifically include：Prepare after above-mentioned transformation Cas9 protein sequences, build improved plasmid vector；Design specific recognition, cutting machin genome and specify site sequence The genome edit tool sgRNA of row, using the method for common microinjection by the genome edit tool sgRNA, described change It is slender that the Cas9mRNA that the plasmid vector of (Ub-i-Cas9) is generated through in-vitro transcription after (WT-Cas9) and transformation before making imports monkey Born of the same parents embryonated egg embryo, it is prominent using single blastomere isolation technics and the singe-cell PCR technical appraisement gene targeting efficiency of optimization, homozygosis Become efficiency.

Hereinafter, with Ubiquitin-i represent Ubquitin-L, Ubquitin-P or Ubquitin-R any one.

Based on CRISPR/Cas9 mediated gene shooting methods, the fusion protein provided using the present invention carries out gene targeting, The problem of gene targeting that the presence of CRISPR/Cas9 technologies can be solved is fitted together to effect.The method is particularly applicable in and struck by we Except machin PINK1, during the vector construction of Parkin and ASPM genes.Our experiment includes Cas9 albumen before 1) transforming Expression vector MLM3613-Cas9,2) Ubiquitin-i-Cas9 protein expression vectors and PINK1, Parkin are carried after transformation With the sgRNA expression vectors of ASPM gene targeting sequences；3) and using the method for microinjection is had altogether the genome is compiled The instrument of collecting including：SgRNA, the plasmid vector of (Ub-i-Cas9) turns through external after (WT-Cas9) before the transformation and transformation Record the Cas9mRNA of generation } import the unicellular embryonated egg embryo of monkey；4) the unicellular of single blastomere isolation technics and optimization is utilized Round pcr identification gene targeting efficiency, homozygous mutation efficiency.

There is important relation due to being fitted together to Cas9 albumen expressed after the generation and injection of effect longer half-life period.It is logical Crossing the method for the present invention can realize while Cas9 cutting efficiencies are not influenceed by shortening Cas9 protein half-lifes to quantify It only plays gene targeting effect to timing controlled in embryonated egg embryonic development early stage, so that chimeric mutational effect is reduced, to efficient The disease model and gene defect repairing and treating for setting up non-human primate homozygous mutation are significant.

One aspect of the present invention can cause Cas9 albumen to be degraded rapidly after playing target practice effect in embryonic cell, so that Reduce the chimeric mutational effect of embryonic development.Especially particularly preferred Ubiquitin-R-Cas9 albumen, homozygosis is practiced shooting prominent Variability improves 3.51 times than wild type Cas9.

Importantly, Ubiquitin-i-Cas9 albumen provided by the present invention can improve embryonic gene target practice homozygosis Property.Especially particularly preferred Ubiquitin-R-Cas9 albumen, it is to wild type Cas9 albumen embryo's gene targeting homozygosity Efficiency improves 350%.This is outside technical staff is expected.

Compared with prior art, importance of the invention is：

First, the invention discloses a kind of transformation of Cas9 protein sequences for signal of specifically being degraded containing Ubiquitin load Body and its application in primate machin, experiment prove the improved Cas9 can to PINK1, Parkin and ASPM genes carry out effective gene target practice, and target practice rate is up to 73.83%；Also, compared with WT-Cas9 (8.25%), after transformation Ub-R-Cas9 gene targeting homozygous mutation rates be up to 28.97%, its homozygosis target practice mutation rate improves 3.51 than WT-Cas9 Times.

Second, using whole embryo genomic DNA can not Accurate Analysis Cas9 practice shooting after each cell in embryo Four cells or eight cell stages that injection has target practice RNA are isolated single spilting of an egg by chimeric mutational type and mutation rate, the present invention Ball, and more precisely analyze and identify using singe-cell PCR technology different cell mutation situations and the mutation of same embryonic origin Rate, reliably confirms remarkable result of the Ubiquitin-Cas9 fusion proteins in terms of target practice homozygous mutation rate is improved.

3rd, to verify Ub-i-Cas9 albumen used in the present invention because fast degradation ability causes development of fertilized ova extremely Four cells just no longer play target practice effect before the period is earlier, and the present invention will using embryo's single blastomere isolation technics The single blastomere that four cell stages are separated and come is cultivated when development is four cell and carries out singe-cell PCR again, and qualification result enters one Step proves that Ub-i-Cas9 fast degradation can ensure the homogeneity of the genetic modification type in four cell stage later stages.

Ub-i-Cas9 provided by the present invention use can substantially reduce the gene targeting that CRISPR/Cas9 is mediated Chimeric effect, improves its genetic modification precision, be gene defect repairing and treating and be it is efficient set up with homozygous mutation it is big Animal model provides new method, with potential Important Economic value and significance.

Brief description of the drawings

Fig. 1 is the Ub-i-Cas9 structural representations for including different aminoacids degraded signal (black matrix).

Fig. 2 is Ub-i-Cas9 plasmid construction flow charts.

Fig. 3 is expression and half-life period detection of the Ub-i-Cas9 in 293 cells；Wherein, A.Ub-i-Cas9 is in 293 cells In expression；B.WT-Cas9 and UbR-Cas9 half-life period；C. figure B relative quantification is counted.

Fig. 4 is expression of the WT-Cas9 and Ub-R-Cas9 in machin embryo；Wherein, a.WT-Cas9 and Ub-R-Cas9 Expression in machin embryo；B.Ub-R-Cas9 gene targeting PCR identifications and sequencing result in 293 cells.

Fig. 5 is that Parkin gene targeting efficiencies of the WT-Cas9 and Ub-R-Cas9 in machin embryo compares；Wherein, A.WT-Cas9 and Ub-R-Cas9 target practice efficiency statistical forms in machin embryo；B.WT-Cas9 and Ub-R-Cas9 are in machin The PCR representative results practiced shooting in embryo；C.Ub-R-Cas9 practices shooting sequencing result in machin embryo.

Fig. 6 is that target practice result is identified in singe-cell PCR digestion；Injection has WT-Cas9 and Ub-R-Cas9 embryonated egg embryo thin Born of the same parents be developed to four cell stages separated through single blastomere be further cultured for it is separated unicellular after developing into new four cell.

Embodiment

The invention will be further described below, but embodiments of the present invention are not limited to following embodiment introduction, The equivalent change or accommodation that all principles or theory according to the present invention are made are regarded as the category that the present invention is protected.

Invent the ubiquitin protein degraded signal Ubiquitin-R-myc sequences such as SEQ ID NO.3 being related to；Ubiquitin- R sequences such as SEQ ID NO.4；Myc sequences such as SEQ ID NO.5；Cas9 protein sequences such as SEQ ID NO.6.

The coded sequence such as SEQ ID of the Ubquitin-R-Cas9 fusion proteins of the present invention are encoded in the particular embodiment NO.2. and Ubquitin-L-Cas9, Ubquitin-P-Cas9 be then on SEQ ID NO.2. the 229th~231 cgc difference Replace with cuc, ccc.

Embodiment 1

Build purpose plasmid Ub-i-Cas9 (i represents R, L, the P) expression vector for including different protein degradation signals, signal Figure such as Fig. 1, the structure flow and method is following (Fig. 2).

For ease of building, first with MLM3613Cas9 (Addgene, Plasmid#42251, referred to as WT-Cas9) skeleton matter Two restriction enzyme sites of NotI and EcoRI on grain will contain T7 promoters, NcoI clones restriction enzyme site, translation initiation site and portion (Fig. 2 a, b), pGEX4T1-NotI-T7-NcoI- are connected on pGEX4T-1 plasmids after the segment cut for dividing Cas9 protein sequences Cas9-EcoRI-.Then, design carries the sense primer of NotI restriction enzyme sites and T7 promoter sequences I and marked with Myc- (sequence is respectively such as SEQ ID NO.22, SEQ ID for the anti-sense primer of label sequence, L/P/L sites and NocI restriction enzyme sites Shown in NO.23-25), introduce the DNA sequence dna of L/P/R amino acid simultaneously in anti-sense primer, amplified from HEK293 cell cDNAs Ubiquitin sequences；, T7-Ubiquitin-L/P/R-myc (is expanded into) sequence in people's HEK293 cell cDNAs and passes through NotI PGEX4T1-NotI-T7-NcoI-Cas9-EcoRI- carriers, such as Fig. 2 c, 2d are connected to NcoI clone's restriction enzyme sites.Again will NotI-T7-ubiquitin-L/P/R-Myc tag-Cas9-EcoRI- fragments are connected to NotI and the digestions of EcoRI two position The MLM3613-Cas9 skeleton plasmids of point Linear, successfully construct Ubquitin-R/L/P-Myc tag-Cas9 expression vectors (Fig. 2 e).

SEQ ID NO.22：Ub 5’-1Not1:5’-ATCCGCGGCCGC TAA TAC GAC TCA CTA TAGGGCCATGCAGATCTTCGTGAAGAC-3’T7promoter

3’Primers:

Ub R 3’-204Nco1(SEQ ID NO.23):

5’-ATCCATGGTCTTAGCATGTACCAGATCTTCTTCAGAAATAAG

TTTTTGTTCTTTACCTCGCCCACCTCTGAGACGGAG-3’

Ub L 3’-204Nco1(SEQ ID NO.24):

5’-ATCCATGGTCTTAGCATGTACCAGATCTTCTTCAGAAATAAG

TTTTTGTTCTTTACCTAGCCCACCTCTGAGACGGAG-3’

Ub P 3’-204Nco1(SEQ ID NO.25):

5’-ATCCATGGTCTTAGCATGTACCAGATCTTCTTCAGAAATAAG

TTTTTGTTCTTTACCTGGCCCACCTCTGAGACGGAG-3’

The expression plasmid carrier containing Ub-i-Cas9 built carries out sequencing to its sequence using primer extension method and tested Card, sequencing result, which is demonstrated, to be successfully constructed.

Embodiment 2

Structure contains PINK1, the CRISPR/Cas9 of Parkin and ASPM gene target sequences sgRNA carriers.

The target sequence site of table 1.

Note：Dashed part sequence is sgRNA PAM sequences, i.e. its binding site with target dna sequence.

By above-mentioned constructed sgRNA target sequences carrier and transformation before, improved Cas9 plasmid vectors PmeI enzyme lines Property after glue reclaim, in-vitro transcription synthesis RNA.

Described sgRNA expression vectors, sgRNA expression is controlled containing a T7 promoter, and digestion is being limited with BbsI 20bp sgRNA specific target sequence is can be inserted into after digestion, the recognition site of the specific target sequence is located at outside PINK1 genes second Aobvious son, Parkin gene Second Exons, the extron of ASPM genes the 3rd and the tenth extron.Gene can be carried out in order to screen Edit and the less PINK1 and ASPM genes of effect of missing the target sgRNA sequences, applicant using NCBI Blast softwares in Mi Screened respectively on the PINK1 genes of monkey and the ASPM gene orders of machin effect of missing the target it is low and meet CRISPR/Cas9 knot Close the target sequence site of genome signature (5 '-G (19N)-NGG3 ') or (5 '-CCN (19N)-C-3 '), such as table 1, structure side Method is with reference to existing document (Chang et al., 2013).

Embodiment 3

Ub-i-Cas9 (i represents L P, R) plasma half-life detection of structure.

First, by WT-Cas9 and Ub-i-Cas9 plasmid transfections to 293 cells, after transfecting 48 hours, Western blot Detection Cas9 expressing quantity results are shown, are counted using WT-Cas9 as 100%, Ub-L-Cas9, Ub-P-Cas9, Ub-R-Cas9 points Do not degrade to 84.76%, 80.92% and 63.89%.

Ub-R-Cas9 especially therein, although intersecting only one of which alkali in its sequence with Ub-L-Cas9, Ub-P-Cas9 The difference of base, but it is that its degradation rate is significantly faster than other fusion proteins to have surprisingly found that, has reached very rare journey Degree.

Then, we compare WT-Cas9 and Ub-R-Cas9 half-life period, i.e., after plasmid transfection 24 hours, addition 50 Micromole's cycloheximide is into culture medium, respectively in 0h, 2h, 4h, 8h, 12h, and 24h collects cell, Western blot detection knots Fruit such as Fig. 3 B, shown in C, compared with WT-Cas9, Ub-R-Cas9 protein half-lifes significantly shorten, and about hundred were just degraded at 4 hours / five ten.

Secondly, WT-Cas9 and Ubquitin-R-Cas9 in-vitro transcriptions are synthesized into mRNA microinjections in machin embryo, As shown in table 2, Showed by immune group result Ubquitin-R-Cas9 is more obvious than WT-Cas9 degradation speed in machin embryo Accelerate.Ub-R-Cas9 contents than WT-Cas9 fewer 69.18% in the embryo after injection 48 hours.

Cas9 residual quantities of the table 2.Ubquitin-R-Cas9 with WT-Cas9 in embryo is compared

	Inject residual quantity after 24h	Inject residual quantity after 48h
			Ub-R-Cas9	63.38	18.96
WT-Cas9	100%	61.53

Embodiment 4

Ub-R-Cas9 gene targetings function and Efficiency testing that the success is built.

First, WT-Cas9/Pink2-2sgRNA and Ub-R-Cas9/Pink2-2sgRNA plasmids are transfected to 293 respectively Cell, selects individual cells by dilution process step by step after 48 hours or multiple cells enters performing PCR and identified, through T7E1 detections and Sequencing confirms that Ub-R-Cas9 has normal gene target practice function, such as Fig. 4 b.

Secondly, by WT-Cas9 or Ub-R-Cas9mRNA and sgRNA, microinjection enters monkey one cell embryos altogether.Treat embryo Be developed to four cells or eight cell stages separate single blastomere and carry out singe-cell PCR identification (pcr amplification primer thing sequence is shown in Table 3). In addition, the four cell stages separation for having WT-Cas9 and Ub-R-Cas9 can will be injected using embryo's single blastomere separate mode Single blastomere cultivate again development be four cells when carry out singe-cell PCR, qualification result such as Fig. 6.

The amplimer sequence of table 3.

As a result show：The improved Ub-R-Cas9 carriers can carry out gene to PINK1, Parkin and ASPM genes Practice shooting, target practice rate is up to 73.83%.And Ub-R-Cas9 can significantly improve homozygous gene compared with WT-Cas9 (8.25%) and strike The probability removed is up to 28.97%, improves about 3.5 times, such as Fig. 5.In addition, Fig. 6 results also further demonstrate that Ub-R-Cas9 because of tool The homogeneity for the genetic modification type for having fast degradation ability to ensure for four cell stage later stages, hence it is evident that reduce WT-Cas9 Chimeric effect caused by lasting target practice caused by half-life period is longer.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, All without departing from present disclosure, it is included in protection scope of the present invention.

Claims (14)

1. a kind of Cas9 fusion proteins, including Cas9 fragments and Ubquitin fragments, it is characterised in that described Ubiquitin is Ubiquitin-L, Ubiquitin-P or Ubiquitin-R；

When Ubiquitin is Ubiquitin-R, the amino acid sequence of fusion protein is the amino as shown in SEQ-ID NO.1 Acid sequence；

When Ubiquitin is Ubiquitin-L, the amino acid sequence of fusion protein is the amino as shown in SEQ-ID NO.1 Acid sequence, wherein the 77th replaces with Leucine (leucine)；

When Ubiquitin is Ubiquitin-P, the amino acid sequence of fusion protein is the amino as shown in SEQ-ID NO.1 Acid sequence, wherein the 77th replaces with Proline (proline).

2. fusion protein as claimed in claim 1, it is characterised in that its sequence is the amino acid sequence as shown in SEQIDNO.1.

3. a kind of Cas9 fusion proteins coded sequence, contains Cas9DNA fragments and UbquitinDNA fragments, it is characterised in that institute The UbquitinDNA fragments stated are Ubiquitin-L DNA fragmentations, Ubiquitin-PDNA fragments or Ubiquitin-R DNA Fragment；

When Ubiquitin DNA fragmentations are Ubiquitin-R DNA fragmentations, the DNA sequence dna of fusion protein coding is such as SEQ- DNA sequence dna shown in ID NO.2；

When Ubiquitin DNA fragmentations are Ubiquitin-P DNA fragmentations, the DNA sequence dna of fusion protein coding is such as SEQ- DNA sequence dna shown in ID NO.2, wherein 229-231 replace with ccc；

When Ubiquitin DNA fragmentations are Ubiquitin-L DNA fragmentations, the DNA sequence dna of fusion protein coding is such as SEQ- DNA sequence dna shown in ID NO.2, wherein 229-231 replace with cuc.

4. coded sequence as claimed in claim 3, it is characterised in that its sequence is the DNA sequence dna as shown in SEQIDNO.2.

5. a kind of plasmid containing the sequence as described in claim 3-4 is any.

6. a kind of expression vector containing the sequence as described in claim 3-4 is any.

7. a kind of gene targeting method, it is characterised in that utilize fusion protein or claim as described in claim 1-2 is any Any coded sequences of 3-4 carry out gene targeting, and methods described is not used in the diagnosis or treatment of disease.

8. gene targeting method as claimed in claim 7, it is characterised in that the gene is mammalian genes.

9. gene targeting method as claimed in claim 8, it is characterised in that the gene is non-human mammal gene.

10. gene targeting method as claimed in claim 9, it is characterised in that the gene is non-human primate's base Cause.

11. a kind of reduce the method that zooblast gene targeting is fitted together to effect, it is characterised in that any using such as claim 1-2 The fusion protein or any coded sequences of claim 3-4 carry out gene targeting, and methods described is not used in examining for disease Disconnected or treatment.

12. the method that reduction zooblast gene targeting is fitted together to effect as claimed in claim 11, it is characterised in that described Cell is mammalian cell.

13. the method that reduction zooblast gene targeting is fitted together to effect as claimed in claim 12, it is characterised in that described Cell is non-human mammalian cell.

14. the method that reduction zooblast gene targeting is fitted together to effect as claimed in claim 13, it is characterised in that described Cell is non-human primate's cell.