CN106434748A - Development and applications of heat shock induced Cas9 enzyme transgene danio rerio - Google Patents

Abstract

The invention belongs to the field of biotechnology, and in particular relates to the development and application of a heat-shock-inducible Cas9 enzyme transgenic zebrafish. The present invention firstly provides a Cas9 enzyme expression vector, which uses a heat shock inducible promoter HSP70 to drive downstream Cas9 gene expression. The Cas9 enzyme expression vector and Tol2 mRNA were co-injected into wild-type zebrafish single-cell fertilized eggs, and the heat-shock-inducible Cas9 enzyme transgenic zebrafish was bred, and the gene editing of the CRISPR-Cas9 system in zebrafish was successfully realized. Research, and achieved the knockout of MC4R gene in this transgenic zebrafish for the first time. The Cas9 enzyme expression vector is also suitable for applications such as heat-shock-inducible gene knockout, gene knock-in, and gene expression modification of other fish.

Description

Development and application of a heat shock-inducible Cas9 enzyme transgenic zebrafish

technical field

The invention belongs to the field of biotechnology, and in particular relates to the development and application of a heat-shock-inducible Cas9 enzyme transgenic zebrafish.

Background technique

The zebrafish (Danio rerio) is a bony fish belonging to the genus Danio of the family Cyprinidae in the subclass Actinopterygii, because it has longitudinal dark blue and silver stripes like a zebra on the side of its body. named after the stripes. Zebrafish has a high degree of homology of 87% with human genes, which means that the results obtained using zebrafish as experimental animals can be applied to humans in most cases, so its advantages as a model organism are very prominent. Its juveniles have the characteristics of rapid reproduction, transparent eggs and juveniles, and are often used for monitoring water environment pollutants, making related disease models, and so on. Among them, the maturity of transgenic technology also brings greater prospects for the application of zebrafish. The preparation of transgenic zebrafish usually includes the following steps: First, construct the recombinant expression vector of the transgene, and then introduce the recombinant expression vector with the target gene or fluorescent gene into zebrafish single-cell fertilized eggs by using classical microinjection technology, so that the outer The source target gene or fluorescent gene can be integrated into the chromosome of the zebrafish embryo, and the target gene or fluorescent marker gene can be expressed, and the integration and expression of the foreign gene can be detected by PCR or fluorescent microscope, so as to screen and identify transgenic offspring or transgenic Chimera.

The CRISPR-Cas9 system has been successfully transformed into a third-generation artificial endonuclease, which, like zinc finger endonuclease (ZFN) and transcription activator-like effector nuclease (TALEN), can be used for editing various complex genomes. Its main functional components include gRNA and Cas9 protein that recognize the target site. The gRNA is responsible for recognizing the target site, and the Cas9 protein performs the cutting function. Introduce mutations. At present, this technology has been successfully applied to the precise genome modification of human cells, zebrafish, mice, and bacteria. The types of modification include insertion-deletion mutations, gene-directed knock-in, and large-segment deletions. Due to its high mutation efficiency, simple production and low cost, it is considered to be a molecular tool for targeted genome modification with broad application prospects. MC4R belongs to the family of G-protein-coupled receptors and is a kind of peptide substance secreted by the ventromedial nucleus of the hypothalamus. It plays an important role in regulating energy homeostasis and obesity. Through more than ten years of research, the functional research of MC4R gene in mammals has been relatively thorough. However, due to the lack of effective gene editing methods in fish, the research on the function of MC4R gene in fish has just started.

Contents of the invention

In order to overcome the problems existing in the prior art, the object of the present invention is to provide a production method and application of heat shock-inducible Cas9 enzyme transgenic zebrafish.

In order to achieve the above object and other related objects, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a Cas9 enzyme expression vector, comprising: a Cas9 enzyme expression element and a transposon Tol2 located at both ends of the Cas9 enzyme expression element, and the Cas9 enzyme expression element includes a heat shock inducible promoter HSP70 and Cas9 enzyme gene coding sequence.

Preferably, the heat shock-inducible promoter HSP70 contains the sequence shown in SEQ ID NO.7.

Preferably, the Cas9 enzyme gene coding sequence contains the sequence shown in SEQ ID NO.8.

Preferably, the transposon Tol2 positioned at both ends of the Cas9 enzyme expression element comprises the transposon Tol2 positioned at the 5' end of the Cas9 enzyme expression element and the transposon Tol2 positioned at the 3' end of the Cas9 enzyme expression element.

Preferably, the transposon Tol2 located at the 5' end of the Cas9 enzyme expression element contains the sequence shown in SEQ ID NO.9.

Preferably, the transposon Tol2 located at the 3' end of the Cas9 enzyme expression element contains the sequence shown in SEQ ID NO.10.

Preferably, the Cas9 enzyme expression element also includes a fluorescent marker protein sequence.

Preferably, the fluorescent marker protein sequence is connected to the coding sequence of the Cas9 enzyme gene through a 2A sequence.

Further preferably, the 2A sequence contains the sequence shown in SEQ ID NO.11.

Preferably, the fluorescent marker protein is green fluorescent protein eGFP.

Preferably, the Cas9 enzyme expression vector contains the sequence shown in SEQ ID NO.12.

The second aspect of the present invention provides the use of the aforementioned Cas9 enzyme expression vector in cultivating Cas9 enzyme transgenic animals.

A third aspect of the present invention provides a Cas9 enzyme transgenic zebrafish culture method, comprising: co-injecting the aforementioned Cas9 enzyme expression vector and Tol2 mRNA into wild-type zebrafish single-cell fertilized eggs, and breeding Cas9 enzyme transgenic zebrafish.

Preferably, the Tol2 mRNA contains the sequence shown in SEQ ID NO.13.

Preferably, the injection is by microinjection.

Preferably, the breeding method includes: conducting heat shock induction on the injected fertilized eggs.

Further preferably, the breeding method specifically comprises: incubating the injected fertilized eggs at 28°C for 24 hours, then incubating at 37°C for 1 hour, screening fish eggs with fluorescent markers, and incubating them until they become adult fish.

Preferably, the breeding method also includes the screening of pure line Cas9 enzyme transgenic zebrafish,

Firstly, the chimera P0 expressing fluorescence in fertilized eggs is selected to cross with wild-type zebrafish to generate hybrid F1 generation, and the fluorescent individuals in the offspring are screened; the single fluorescent individual in the F1 generation is tested crossed with the wild type to obtain For the heterozygous F2 generation, screen the fluorescent individuals in the progeny; self-cross the fluorescent female and male fish in the heterozygous F2 generation to obtain the F3 generation; continue to testcross the obtained F3 generation with the wild type and count the hatching of F4 fish eggs Fluorescence status; if all F4 fish eggs are fluorescent, it is determined that the parental F3 is a pure Cas9 enzyme transgenic zebrafish.

The fourth aspect of the present invention provides a Cas9 enzyme transgenic zebrafish obtained by the aforementioned method.

The fifth aspect of the present invention provides the use of the Cas9 enzyme transgenic zebrafish in the study of fish gene function.

The fish gene includes but not limited to MC4R gene.

The sixth aspect of the present invention provides a method for studying fish gene functions, comprising: injecting sgRNA targeting fish genes into single-cell fertilized eggs of pure Cas9 enzyme transgenic zebrafish to knock out fish genes .

The seventh aspect of the present invention provides a CRISPR-Cas9 gene knockout kit, said kit comprising: the aforementioned Cas9 enzyme expression vector and sgRNA.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention first constructs a Cas9 enzyme expression vector, which uses the heat shock inducible promoter HSP70 to drive downstream Cas9 gene expression. The Cas9 enzyme expression vector and Tol2 mRNA were co-injected into wild-type zebrafish single-cell fertilized eggs, and the heat-shock-inducible Cas9 enzyme transgenic zebrafish was bred, and the gene editing research of the CRISPR-Cas9 system in zebrafish was successfully realized. , and achieved the knockout of MC4R gene in this transgenic zebrafish for the first time. The Cas9 enzyme expression vector is also suitable for applications such as heat-shock-inducible gene knockout, gene knock-in, and gene expression modification of other fish.

Description of drawings

Fig. 1 is the map of Cas9 expression vector pTol2-HSP70-Cas9-2A-eGFP of the present invention.

Figure 2 is a map of plasmid pTol2-LoxP-CMV-eCFP.

Figure 3 is a map of plasmid pISceI-HSP70-Cas9-2A-eGFP.

Fig. 4 is a sequence diagram of the insertion and deletion of MC4R gene, and the sequences involved in Fig. 4 are respectively shown in SEQ ID NO.14-17.

Fig. 5 is an electrophoresis diagram of the detection result of MC4R gene T7E1.

detailed description

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by each manufacturer.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology. These techniques have been fully described in the existing literature, specifically MOLECULAR CLONING such as Sambrook: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; theseseries METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. .), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 Obtaining the Cas9 enzyme expression vector pTol2-HSP70-Cas9-2A-eGFP

Use BamHI and SalI to double digest the plasmid vector pTol2-CMV-eGFP (as shown in Figure 2). SalI2.5ul, add water to make up 50ul. The 7993bp fragment was recovered by agarose gel electrophoresis to obtain the backbone fragment pTol2.

The plasmid vector pISceI-HSP70-Cas9-2A-eGFP was double digested with BamHI and SalI (as shown in Figure 3). ug/ul), BamHI 2.5ul, SalI 2.5ul, add water to make up 50ul. After the digestion was completed, the 6969bp fragment was recovered by agarose gel electrophoresis to obtain the insert fragment HSP70-Cas9-2A-eGFP.

The backbone fragment pTol2 and the insert fragment HSP70-Cas9-2A-eGFP were ligated. The ligation reaction system: HSP70-Cas9-2A-eGFP 3ul, pTol2 2ul, 10xBuffer 2ul, T4 ligase 0.2ul, sterile water 12.8ul. T4 DNA ligase was used to ligate overnight at 16°C.

Take 10ul of the ligation product and transform into 90ul Escherichia coli competent cells DH5α, gently pipette and mix well, incubate on ice for 30min, heat shock at 42°C for 90s, immediately place on ice for 2min, add 900ul of LB medium at 37°C , activated on a shaker at 180rpm at 37°C for 1h, centrifuged at 5000rpm for 3min to enrich the activated bacteria solution, sucked off 900ul of the supernatant, mixed 100ul of the bacteria solution and spread it on an ampicillin-resistant culture dish, and cultured it upside down in a 37°C incubator overnight . Randomly select 10 single colonies for detection by PCR method, expand the positive clones in 6ml LB liquid medium, culture overnight at 37°C and 250rpm, collect the bacterial liquid and extract the plasmid. The obtained plasmid was sequenced and verified, and the sequencing results were consistent with expectations, and the Cas9 enzyme expression vector pTol2-HSP70-Cas9-2A-eGFP was obtained.

As shown in Figure 1, the Cas9 enzyme expression vector pTol2-HSP70-Cas9-2A-eGFP includes: the Cas9 enzyme expression element and the transposon Tol2 positioned at both ends of the Cas9 enzyme expression element, and the Cas9 enzyme expression element includes heat shock Inducible promoter HSP70, and Cas9 enzyme gene coding sequence.

Wherein, the heat shock inducible promoter HSP70 contains the sequence shown in SEQ ID NO.7. For the sequence shown in SEQ ID NO.7, please refer to the sequence listing section for details.

Wherein, the Cas9 enzyme gene coding sequence contains the sequence shown in SEQ ID NO.8. For the sequence shown in SEQ ID NO.8, please refer to the sequence listing section for details.

The transposon Tol2 located at both ends of the Cas9 enzyme expression element comprises the transposon Tol2 located at the 5' end of the Cas9 enzyme expression element and the transposon Tol2 located at the 3' end of the Cas9 enzyme expression element.

Wherein, the transposon Tol2 located at the 5' end of the Cas9 enzyme expression element contains the sequence shown in SEQ ID NO.9. For the sequence shown in SEQ ID NO.9, please refer to the sequence listing section for details.

The transposon Tol2 located at the 3' end of the Cas9 enzyme expression element contains the sequence shown in SEQ ID NO.10. For the sequence shown in SEQ ID NO.10, please refer to the sequence listing section for details.

The Cas9 enzyme expression element also includes a fluorescent marker protein sequence. The fluorescent marker protein sequence is connected with the coding sequence of the Cas9 enzyme gene through the 2A sequence. The 2A sequence contains the sequence shown in SEQ ID NO.11. For the sequence shown in SEQ ID NO.11, please refer to the sequence listing for details.

The Cas9 enzyme expression vector pTol2-HSP70-Cas9-2A-eGFP contains the sequence shown in SEQ ID NO.12. For the sequence shown in SEQ ID NO.12, please refer to the sequence listing section for details.

Example 2 Injection of Cas9 enzyme expression vector into zebrafish fertilized eggs

(1) Obtaining zebrafish fertilized eggs

Before fertilization, the male and female broodstock were separated and placed in the spawning box at a ratio of 1:1-2 for isolation culture. The egg-laying box was placed in a constant temperature environment of 26°C-29°C for overnight culture in the dark, and the photoperiod was 14h in daylight and 10h in darkness. At the end of the cultivation, the partition board was removed and the inner layer with the fence at the bottom was obliquely placed on the outer layer mating box, and placed in a constant temperature environment to obtain fertilized zebrafish eggs.

(2) Inject the Cas9 enzyme expression vector into zebrafish fertilized eggs

Using a microinjector and referring to the microinjection method, the Cas9 enzyme expression vector pTol2-HSP70-Cas9-2A-eGFP constructed in Example 1 (as shown in Figure 1) and Tol2mRNA were co-injected into the obtained zebrafish single-cell fertilization egg. The injection system is 10ul, in which Cas9 expression vector is 50ng/ul, Tol2mRNA 1ul, phenol red 2ul, add water to make up 10ul. Take 0.6ul injection solution and inject it into the capillary, put it into the injector, and inject it into the animal poles of the zebrafish fertilized eggs in sequence. The entire operation process was completed within 45 minutes to ensure that the injection was completed before the first cleavage of the cells.

Wherein, the Tol2 mRNA contains the sequence shown in SEQ ID NO.13. For the sequence shown in SEQ ID NO.13, please refer to the sequence listing section for details.

(3) Fluorescence screening and offspring cultivation

After the injection, the fertilized eggs were cultured at 28°C, and after 24 hours, they were incubated in a constant temperature water bath at 37°C for 1 hour, then observed with a stereo microscope, and the eggs with green fluorescence were screened and incubated until they reached adult fish. On the 1d-4d after screening, the zebrafish embryos were cultivated in a constant temperature environment of 26°C-29°C to obtain juveniles; on the 5th-9d, the juveniles were fed with yolk water twice a day; Feed Artemia worms and yolk water ground with a filter. The specific method is to put a small amount of Artemia worms into the breeding container first, then add the yolk water ground with a filter after 25-35 minutes, and feed twice a day; after the 17th day, the young The fish were fed with Artemia, 2 times a day, and the fish water was changed once a day.

(4) Screening of stable genetic pure line Cas9 enzyme transgenic zebrafish

First, the caudal fin of the 2-month-old P0 chimera was cut to extract genomic DNA, and the Cas9 gene sequence was detected by PCR. The PCR program was: 94°C for 5 minutes; 94°C for 5s, 55°C for 15s, 72°C for 20s, and 72°C for 5 minutes. Screen the P0 zebrafish with positive PCR results and hybridize with the wild-type zebrafish, incubate the eggs in a 37°C water bath for 1 hour, then observe the fluorescence under a stereofluorescence microscope, and select eggs with green fluorescence to grow to sexual maturity , get the hybrid F1 generation. The F1 generation was detected by PCR, and the zebrafish with positive PCR results were selected and cultivated to sexual maturity, and a single individual in the hybrid F1 was tested with the wild type. After two months of age, positive zebrafish were screened by PCR and cultivated to sexual maturity. This is hybrid F2. Since the heterozygous F2 originates from the same parent (heterozygous F1 and wild type), all F2 heterozygotes have the same genotype. The female fish in the heterozygous F2 was selfed with the male fish, and among the offspring F3, there was a 1/4 probability of obtaining a homozygous F3 containing the Cas9 transgenic zebrafish. After two months of age, all homozygotes and heterozygotes were screened by PCR pruning detection. In order to verify the homozygous F3, the screened F3 was tested with the wild type and PCR was used to detect the F4 egg DNA. If all the F4 eggs were If the PCR is positive, and the fish eggs are incubated at 37°C and then observed with a stereoscopic fluorescence microscope, they all have green fluorescence, then it can be determined that the parent F3 is a pure line.

The knockout of embodiment 3 zebrafish MC4R gene

(1) Determination of MC4R gene targeting sites and preparation of gRNA

Design the MC4R gene targeting site, select the appropriate targeting sequence according to the results given by the website (http://zifit.partners.org/favicon.ico), the targeting sequence selected by the present invention is shown in SEQ ID NO.1, specifically For: GGGGGTGTTTGTGGTGTGCT.

The present invention adopts the method of PCR to amplify the transcription template of gRNA. First, design primers according to the selected targeting sequence, the upstream primer sequence is as SEQ ID NO.2, specifically:

TAATACGACTCACTATAGGGGGTGTTTGTGGTGTGCTGTTTTAGGCTAGAAATAGC; the downstream primer sequence is shown in SEQ ID NO.3, specifically: AGCACCGACTCGGTGCCAC.

The plasmid containing the gRNA backbone is used as a template, and the backbone sequence is shown in SEQ ID NO.4, specifically: AGCTTGAAATTAATACGACTCACTATAGGGAGTCTTCAGATCTAACACAACTCGAGGAAGACATGTTTTGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAGTGGCACCGAGTC GGTGCG.

The PCR amplification program was: 98°C for 5min; 98°C for 10s, 58°C for 30s, 72°C for 20s, a total of 35 cycles, and 72°C for 5min. After the PCR product was detected by 1% agarose gel electrophoresis, the product was recovered by a PCR product purification kit. The purified product was used as a template for in vitro transcription. The transcription system was 20ul, including 600ng of purified product, 1ul of T7 transcriptase, 2ul of reaction buffer, 1ul of dNTP mixture, and 20ul of sterile water. Incubate at 37°C for 3h. After the transcript was detected by 1% agarose gel electrophoresis, it was purified and recovered by an RNA purification kit to obtain gRNA, which was stored in a -80°C refrigerator.

(2) In vitro microinjection and knockout verification

Prepare fertilized eggs: select sexually mature and stable genetic pure-line Cas9 transgenic zebrafish (see Example 2 for details), 2 female fish and 1 male fish, put them into the spawning box, and insert a transparent partition in the middle to separate the male and female fish. Place the egg-laying box in a constant temperature room at 26°C-29°C for overnight rearing in the dark, with a photoperiod of 14 hours in the daytime and 10 hours in the dark. Microinjection. In the morning of the next day, pull out the transparent partition, keep a quiet environment, and let the male and female fish chase and lay eggs by themselves. Immediately after spawning, the fertilized eggs were collected and injected with a microinjector. The injection system is: gRNA 30ng/ul, phenol red 0.5ul, sterile water to supplement 10ul. The injection site is the animal pole of the fertilized egg, and the whole operation process is completed within 45 minutes to ensure that the injection is completed before the first cleavage of the cells. Knockout validation. The injected zygotes were cultured at 28°C. After 24 hours, take 10 fertilized zebrafish eggs from the control group and 10 fertilized zebrafish eggs from each of the two experimental groups, extract DNA respectively, and design primers with a sequence of about 500 bp on both sides of the gRNA targeting site. The primer sequence is as shown in SEQ ID NO.5 and shown in SEQ ID NO.6,

SEQ ID NO.5: GACCGCTACATCACAATCT;

SEQ ID NO. 6: TTGGCTTCTGAAGGCATAT.

Zebrafish DNA was amplified with these primers, 98°C for 5min; 98°C for 10s, 52°C for 20s, 72°C for 20s, a total of 35 cycles, 72°C for 5min. T7 endonuclease I (T7E1) has the characteristics of recognizing and cutting incompletely paired DNA and heteroduplex DNA, so this enzyme is used for mutation verification. The reaction system is: PCR product 8.5ul, buffer 1ul, after mixing evenly, Annealed in a PCR machine. The annealing program is: 95°C for 5min, 94°C for 2sec, -0.1°C/cycle, 200times, 75°C for 1sec, -0.1°C/cycle, 600times, 16°C for 2min. After the annealing is completed, add 0.5ul T7E1 enzyme and incubate at 37°C for 30min. After the incubation was completed, 2% agarose gel electrophoresis was used to detect whether the knockout was successful, and the results are shown in FIG. 5 . Then subcloned into the sequencing vector, sent for sequencing and compared with the wild-type gene sequence to confirm the effective knockout of the target gene. The fish (F0) with successful tail-cut detection were crossed with the wild type to obtain the F1 generation heterozygote, and compared with the wild type by sequencing, the genotypes with 5, 12 and 8 bases missing were respectively obtained, which caused the frameshift mutation of the gene . The result is shown in Figure 4.

The Cas9 transgenic zebrafish can effectively knock out the MC4R gene, indicating that the Cas9 transgenic zebrafish was successfully constructed. Compared with modern technology, the beneficial effects of the present invention are: (1) the implementation process of the method is simple and easy to control; (2) the method can obtain Cas9 transgenic zebrafish; (3) the obtained zebrafish strain can provide convenience for gene knockout; (4) Effective knockout of the MC4R gene was achieved.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any form and in essence. Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those who are familiar with this profession, without departing from the spirit and scope of the present invention, when they can use the technical content disclosed above to make some changes, modifications and equivalent changes of evolution, are all included in the present invention. Equivalent embodiments; at the same time, all changes, modifications and evolutions of any equivalent changes made to the above-mentioned embodiments according to the substantive technology of the present invention still belong to the scope of the technical solution of the present invention.

Claims (10)

1. a kind of Cas9 expression of enzymes carrier, including：Cas9 expression of enzymes element and be located at described Cas9 expression of enzymes element two ends Transposon Tol2, described Cas9 expression of enzymes element includes heat-shock inducible promoter HSP70 and Cas9 enzyme gene coded sequence.

2. Cas9 expression of enzymes carrier according to claim 1 is it is characterised in that be located at described Cas9 expression of enzymes element two ends Transposon Tol2 include the transposon Tol2 that holds positioned at described Cas9 expression of enzymes element 5 ' and be located at described Cas9 expression of enzymes unit The transposon Tol2 that part 3 ' is held.

3. Cas9 expression of enzymes carrier according to claim 1 it is characterised in that described Cas9 expression of enzymes element also include glimmering Signal protein sequence, described fluorescent marker protein sequence is connected with Cas9 enzyme gene coded sequence by 2A sequence.

4. Cas9 expression of enzymes carrier according to claim 1 it is characterised in that described Cas9 expression of enzymes carrier contain as Sequence shown in SEQ ID NO.12.

5. as described in Claims 1 to 4 any claim Cas9 expression of enzymes carrier cultivate Cas9 enzyme transgenic animal in Purposes.

6. a kind of Cas9 enzyme transgenic zebrafish cultural method, including：Will be as described in Claims 1 to 4 any claim Cas9 expression of enzymes carrier and Tol2mRNA co-injection enter the unicellular germ cell of wild-type zebrafish, and selection-breeding obtains Cas9 enzyme and turns base Because of Brachydanio rerio.

7. method according to claim 6 is it is characterised in that described selection includes：Germ cell after injection is entered Row heat-inducible, specifically includes：Germ cell after injection cultivates 24h under the conditions of 28 DEG C, is then incubated 1h under the conditions of 37 DEG C, Screening is with fluorescently-labeled fish roe incubation culture to adult fish.

8. method according to claim 6 is it is characterised in that described selection also includes being sheerly Cas9 enzyme transgenic speckle Express the chimera P0 of fluorescence in the screening of horse fish, first selective fertilization ovum and wild-type zebrafish carries out hybridization and produces heterozygote F1 generation, fluoresce in screening offspring individuality；By the single individual and wild type fluorescing in described F1 generation carry out test cross obtain miscellaneous In fit F2 generation, fluoresce in screening offspring individuality；By the raun fluorescing in heterozygote F2 generation and milter selfing, obtain F3 generation； Gained F3 with wild type test cross and is counted the situation that fluoresces after the incubation of F4 fish roe for single individual continuation, if F4 fish roe is all Fluoresce it is determined that its parent F3 is pure lines Cas9 enzyme transgenic zebrafish.

9. fish gene is used for by the Cas9 enzyme transgenic zebrafish that claim 6～8 any claim methods described obtains Purposes in the research of function.

10. a kind of method of researching fish gene function, including：SgRNA for fish gene is expelled to by claim 6 In the unicellular germ cell of Cas9 enzyme transgenic zebrafish that～8 any claim methods describeds obtain, to knock out Brachydanio rerio Corresponding gene.