CN110904103A - Zebra fish mutant with GRNa gene knockout function and preparation method thereof - Google Patents
Zebra fish mutant with GRNa gene knockout function and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a GRNa knockout zebra fish strain, which comprises the steps of screening sgRNA of a targeted GRNa gene, constructing an eukaryotic expression vector carrying Cas9 and sgRNA expression elements, screening positive zebra fish founder, screening second-generation daughter, identifying the expression condition of the GRNa gene, and proving that the obtained GRNa knockout zebra fish strain can be normally mated and spawned and can be stably transmitted to offspring through more than three generations of mutual internal crossing. And confirmed at the genomic, mRNA, protein level, confirming that GRNa gene is hardly expressed. Can be used as a tool for researching the zfGRNa gene. Relevant experiments such as HE dyeing, immunohistochemistry and the like prove that the GRNa gene knockout zebra fish product system shows an obvious pathological phenotype, can be used for researching a mechanism of GRNa in treatment of nervous system diseases, metabolic diseases and other diseases and relevant drug screening, can also be used for research of zebra fish science and application in economic culture, and has good commercial value.
Description
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to a GRNa gene knockout zebra fish mutant and a preparation method thereof, which can be used as a tool for GRNa gene function research and related drug screening.
Background
Progranulin (PGRN) is a multifunctional growth factor, and heterozygous deletion of this gene is associated with frontotemporal dementia (FTLD), whereas homozygous deletion is considered a causative factor of ncl (neural ceroid lipofuscinosis). The gene has been studied extensively in human disease and mouse models, but very little has been studied about it in zebrafish. The zebra fish has the biological characteristics of fast growth and development, short breeding period, large number of offspring, in-vitro fertilization, embryo transparency and the like, and is very suitable for gene editing.
There are four homologues for this gene in zebrafish, GRNa, GRNb, GRN-1, GRN-2. Research shows that when the morphholino technology is used for inhibiting the expression of GRNa protein, zebra fish shows remarkable pathological changes such as liver damage, myoblast apoptosis, motor neuron damage and the like. However, these pathological phenomena have yet to be further confirmed due to the toxic side effects of the morpholino technology itself. Therefore, the method establishes a stably inherited GRNa gene knockout zebra fish strain to research the function of the GRNa gene, deeply explores the connection and generation mechanism of the GRNa gene and neurodegenerative diseases, provides a stable and reliable model organism for screening targeted drugs, and undoubtedly has important significance.
Gene-trap, ZFN, TALEN and the like are commonly used in the prior gene knockout means, but the technologies are complex to operate, long in period and expensive. The gene knockout model established based on the CRISPR-Cas9 system has the characteristics of high efficiency, quickness, easiness in mastering and the like, is widely applied at present, and is considered to be a genome editing tool with wide prospect.
Disclosure of Invention
The invention aims to provide a preparation method of a GRNa gene knockout zebra fish strain, which utilizes a CRISPR-cas9 technology to randomly cut a DNA sequence at a target site, and causes frame shift mutation through self repair of cells to obtain zebra fish GRNa gene double allele which is completely knocked out.
In order to achieve the purpose, the invention adopts the following technical solutions:
a preparation method of a GRNa gene knockout zebra fish strain is characterized by comprising the following steps:
(1) sgRNA for screening targeted GRNa gene
Searching a genome sequence of a zebra fish GRNa gene from NCBI, selecting five sgRNA binding sites in an exon region of a coded protein by utilizing sgRNA design software, designing corresponding sgRNA primers, annealing the sgRNA primers, and connecting the sgRNA primers to a sgRNA expression vector;
(2) preparation of sgRNA and Cas9mRNA of zebra fish GRNa gene
Obtaining sgRNA and Cas9mRNA of the GRNa gene of the zebra fish by in vitro transcription by using an in vitro transcription kit;
(3) microinjection of sgRNA and Cas9mRNA into zebra fish fertilized egg single cell stage
The male and female zebra fish are respectively placed into a mating tank at night before microinjection and separated by a baffle plate, the baffle plate is pulled out in the morning next day, the male and female zebra fish are mated, fertilized eggs are collected, and Cas9mRNA and sgRNA mRNA of a target gene GRNa are injected into the fertilized eggs under a microscope within half an hour;
(4) target efficiency detection
After microinjection is carried out for 24 hours, each sgRNA and Cas9 injection embryo 4 tubes are collected, five eggs in each tube are extracted, genome DNA is extracted, PCR amplification is carried out on a targeting region, and the sgRNA with the highest cutting activity is screened by a T7E1 method;
(5) culturing the embryo with the targeting effect to adult fish, and performing internal crossing to lay eggs; randomly taking tube embryos, taking 5 eggs per tube, extracting a genome, carrying out PCR amplification on a targeting area, and carrying out enzyme digestion on T7E1 to identify whether targeting exists;
(6) culturing embryos with the targeting effect to adult fishes, cutting tails one by one to extract genomes, carrying out PCR amplification on targeting areas, sending the amplified target areas to a company for sequencing to see whether base insertion or deletion exists and frame shift mutation or nonsense mutation is caused, and thus effective targeting is achieved;
(7) breeding the heterozygote or homozygote zebra fish which are effectively targeted to adult fish, and mating and spawning to obtain the next generation of zebra fish; determining the F0 generation of the zebra fish mutant by screening, respectively hybridizing the F0 generation mutant with wild zebra fish to obtain F1 generation embryos, culturing at 28 ℃, and observing the survival rate of the F1 generation embryos at the initial stage; after fertilization for two days, 5 embryos are respectively taken from each mutant F1 generation for mutation inheritance identification; extracting genome of each embryo separately, PCR amplifying the region near the target site, sending to company for sequencing and identification to determine whether the mutation can be inherited to F1 generation;
(8) breeding the zebrafish mutant in the F1 generation to adult fish if the presence of the sense mutation is detected from the F1 generation embryo; respectively carrying out tail shearing on each F1 generation adult zebra fish, and screening F1 generation mutants;
(9) hybridizing female fishes and male fishes in the F1 generation mutant to obtain F2 generation embryos, culturing the F2 generation embryos at 28 ℃, observing the development condition of the embryos, and knocking out genes to prevent the embryos from dying if the embryos are normally developed; culturing F2 embryo to adult fish, cutting tail, extracting genome, PCR sequencing, and identifying genotype to see whether homozygote exists; carrying out subsequent experimental study on heterozygote or homozygote zebra fish after the genotype identification;
(10) and hybridizing the F2 generation mutant homozygote adult fish female fish and male fish to obtain F3 generation, namely the GRNa gene knockout zebra fish strain.
According to the invention, the five sgRNA sites are respectively:
SgRNA1：TTTGTCCATTGCTGTCCTA；
SgRNA2：TTATGTACCGAAGAGCCCT；
SgRNA3：CTACTACTCCCAATGTGAT；
SgRNA4：AAATGTGACGTAGCTGCG；
SgRNA5：GTGCCCGTCCGTCCAATC。
according to the preparation method of the zebra fish strain with the GRNa gene knockout, the CRISPR/Cas9 technology is utilized to successfully destroy the GRNa gene of the zebra fish, and the obtained zebra fish strain with the GRNa gene knockout is subjected to intercross for more than three generations, so that the fact that the zebra fish strain with the GRNa gene knockout can be normally mated and spawned, and the GRNa gene knockout can be stably inherited to filial generations. And confirmed at the genomic, mRNA, protein level, confirming that GRNa gene is hardly expressed. Can be used as a tool for researching the zfGRNa gene. Relevant experiments such as HE staining and immunohistochemistry prove that the GRNa gene knockout zebra fish product line shows an obvious pathological phenotype, can be used for researching the mechanism of GRNa in diseases such as treatment of nervous system diseases and metabolic diseases and related drug screening, can also be used for the application of zebra fish research and economic breeding, and has good commercial value.
Drawings
FIG. 1 is a schematic diagram of GRNa gene and sgRNA design sites;
FIG. 2 is an electrophoresis diagram of the cleavage identification of microinjected embryo GRNA target site T7E1 (in the figure, A: foundry, B: F1 generation, C: F2 generation);
FIG. 3 is a graph of the sequencing results of PCR fragments of the GRNa sgRNA targeting region;
FIG. 4 is a histogram of mRNA expression levels of GRNa in the qPCR assay second generation F2 pure and mutant;
FIG. 5 is a graph showing the homozygous mutant of GRNa knockout zebra fish and the change of body weight and body length. Section HE staining pattern.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Detailed Description
Research shows that GRNa gene is involved in growth, damage repair, inflammation, nerve retrogression and other physiological and pathological processes. The research of the applicant finds that the zebra fish with the GRNa gene deletion shows an obvious pathological phenotype, and can be used for researching the GRNa gene function and used as a tool for screening related medicines.
The embodiment provides a preparation method of a GRNa gene knockout zebra fish strain, which comprises the following steps:
(1) sgRNA for screening targeted GRNa gene
Searching a genome sequence of a zebra fish GRNa gene from NCBI, selecting five sgRNA binding sites (figure 1) in an exon region of a coded protein by utilizing sgRNA design software (https:// crispr. cos. uni-heidelberg. de /), designing corresponding sgRNA primers, annealing the sgRNA primers and connecting the sgRNA primers to a sgRNA expression vector;
the five sgRNA loci are respectively as follows:
SgRNA1：TTTGTCCATTGCTGTCCTA；
SgRNA2：TTATGTACCGAAGAGCCCT；
SgRNA3：CTACTACTCCCAATGTGAT；
SgRNA4：AAATGTGACGTAGCTGCG；
SgRNA5：GTGCCCGTCCGTCCAATC。
(2) preparation of sgRNA and Cas9mRNA of zebra fish GRNa gene
Obtaining sgRNA and Cas9mRNA of zebra fish GRNa gene by in vitro transcription with an in vitro transcription kit (Life Technology, AM 1345);
(3) microinjection of sgRNA and Cas9mRNA into zebra fish fertilized egg single cell stage
And (3) respectively placing the male and female zebra fish into a mating tank at night before microinjection, separating the male and female zebra fish by using a baffle plate, pulling out the baffle plate in the morning next day, mating the male and female zebra fish, collecting fertilized eggs, and injecting Cas9mRNA and sgRNA mRNA of the target gene GRNa into the fertilized eggs under a microscope within half an hour.
(4) Target efficiency detection
After microinjection is carried out for 24 hours, each sgRNA and Cas9 are collected and injected into 4 tubes of embryos, five eggs in each tube are extracted, genome DNA is extracted, a targeting region is subjected to PCR amplification, and the sgRNA with the highest cleavage activity is screened by a T7E1 method.
(5) Culturing the embryo with the targeting effect to adult fish, and performing internal crossing to lay eggs. Randomly taking tube embryos, taking 5 eggs per tube, extracting a genome, carrying out PCR amplification on a targeting region, and carrying out enzyme digestion on T7E1 to identify whether targeting exists.
(6) Culturing embryos with the targeting effect to adult fishes, cutting tails one by one to extract genomes, carrying out PCR amplification on the targeting area, sending the amplified target area to a company for sequencing to see whether base insertion or deletion exists and cause frameshift mutation or nonsense mutation, and the method is effective targeting.
(7) And breeding the heterozygote or homozygote zebra fish which are effectively targeted to adult fish, and mating and spawning to obtain the next generation of zebra fish. Determining zebra fish mutant F0 generation through a series of previous screens, respectively hybridizing the mutant F0 generation with wild zebra fish to obtain F1 generation embryo, culturing at 28 ℃, and observing the survival rate of F1 generation at the initial stage; after fertilization for two days, 5 embryos are respectively taken from each mutant F1 generation for mutation inheritance identification; extracting genome of each embryo separately, PCR amplifying the region near the target site, sending to company for sequencing and identification to determine whether the mutation can be inherited to F1 generation;
(8) breeding the zebrafish mutant in the F1 generation to adult fish if the presence of the sense mutation is detected from the F1 generation embryo; respectively carrying out tail shearing on each F1 generation adult zebra fish, and screening F1 generation mutants;
(9) and hybridizing female fishes and male fishes in the F1 generation mutant to obtain F2 generation, culturing embryos at 28 ℃, observing the development condition of the embryos, and knocking out genes to prevent the embryos from dying if the embryos are normally developed. Culturing F2 embryo to adult fish, cutting tail, extracting genome, PCR sequencing, and identifying genotype to see if there is homozygote. After the genotype identification, the heterozygote zebra fish or the homozygote zebra fish can be subjected to subsequent experimental study.
(10) And hybridizing the F2 generation mutant homozygote adult fish female fish and male fish to obtain F3 generation, namely the GRNa gene knockout zebra fish strain.
The following is a specific example given by the inventors, which is merely for better understanding of the present invention and the present invention is not limited to the example.
Example (b):
(1) the zfGRNa sgRNA linker was added with the base of the digested sticky end to facilitate ligation into an expression vector, and primers were synthesized by huada gene corporation, the sequences of which are shown in the following table:
annealing each pair of sgRNA fragments at room temperature and connecting the sgRNA fragments into a pT7/sgRNA backbone vector linearized by BbsI enzyme digestion to obtain a pT7/zfGRNa sgRNA expression vector.
(2) Preparation of sgRNA and Cas9mRNA of zebra fish GRNa gene
The purchased pT7-Pt3ts-nCas9n plasmid (commercially available addrene, #64237) was linearized with SpeI enzyme in the following reaction system:
reaction conditions are as follows: at 37 ℃ for 2 hours.
mu.L of the enzyme-cleaved product was added to 2. mu.L of 10 XDNA Loading Dye and 16. mu.L of DW, and the sample and DL15000 DNAmarker were added to the well of the nucleic acid gel spot, and electrophoresed under a voltage of 160V, and a band of 9kbp was observed in the nucleic acid gel imaging system.
After the plasmid was completely linearized, the fragment was recovered using a gel recovery kit from AXYGEN, and dissolved in DEPC water without rnase. Using Life Technology, mMESSAGE
The purified pT7-Pt3ts-nCas9n linearized fragment obtained in the previous step was transcribed in vitro by the T7 Ultra Kit (AM 1345).
(3) Microinjection of sgRNA and Cas9mRNA into zebra fish fertilized egg single cell stage
And (3) respectively placing the male and female zebra fish into a mating tank at night before microinjection, separating the male and female zebra fish by using a baffle plate, pulling out the baffle plate in the morning next day, mating the male and female zebra fish, collecting fertilized eggs, and injecting the Cas9mRNA and the target gene sgRNA mRNA into the fertilized eggs under a microscope within half an hour.
(4) Targeting efficiency detection sgRNA targeting activity screening
After microinjection for 24 hours, each sgRNA and Cas9 were collected and injected into 4 tubes of embryos, five eggs per tube were added with 20ul of 50mM NaOH solution, at 95 ℃ for 20min, and 2ul of 1M Tris-Hcl (pH 8.0) was added for neutralization to extract genomic DNA. The targeting region was PCR amplified, and the sgrnas with the highest cleavage activity were screened by analyzing the cleavage efficiency of each sgRNA by T7E1 assay. In this example, sgRNA4 was selected as the sgRNA to be used finally.
The T7E1 assay procedure is as follows: using the extracted genome DNA as a template to amplify DNA fragments which are near three cutting sites of the target zfGRNa sgRNA4, wherein the primer sequences are as follows:
and (3) performing denaturation annealing on the PCR product obtained by amplification, and recovering and purifying by using agarose gel electrophoresis. 500ng of the purified PCR product was treated with 0.5. mu. l T7E1 enzyme at 37 ℃ for 25 minutes and detected by agarose gel electrophoresis. The detection result is shown in figure 2.
(5) Culturing the embryo with the targeting effect to adult fish, and performing internal crossing to lay eggs. Randomly taking 4 tubes of embryos, taking 5 eggs per tube, extracting a genome, carrying out PCR amplification on a targeting region, and carrying out enzyme digestion on T7E1 to identify whether targeting exists. The results of the gene targeting efficiency test are shown in FIG. 2.
(6) Culturing embryos with targeting effects to adult fishes, shearing tail fins one by one, extracting genomes by utilizing a Tiangen kit and according to the kit specification, carrying out PCR amplification on targeting regions, directly sending PCR products to Huada company for sequencing to see whether base insertion or deletion exists or not, and enabling the number of the inserted or deleted bases to cause frameshift mutation or nonsense mutation, so that effective targeting is achieved. The sequencing results are shown in FIG. 3.
(6) And culturing the heterozygote or homozygote zebra fish which are effectively targeted for three months to grow into adult fish, and mutually mating and spawning to obtain the next generation of zebra fish. Subsequent experimental studies can be performed to identify the correct heterozygous or homozygous zebrafish.
(7) Identification of mRNA of zebra fish line with GRNa gene knockout function
And (3) taking wild zebra fish and GRNa gene knockout zebra fish, and extracting the total RNA of the tissue according to the operation of the instruction of the total RNA extraction kit of the tissue. And reverse transcription into cDNA was performed according to the instructions of the reverse transcription kit. As a real-time quantitative PCR template, primers were designed and the target gene was amplified, 3 times for each reaction with GAPDH as an internal control. Changes in GRNa mRNA expression levels of GRNa knockout zebrafish were detected by comparison with zfGRNa mRNA expression levels of control cells. The detection results are shown in figure 4.
Detecting the expression level of the GRNa gene of the zebra fish by an RT-PCR method by using the following pair of primers:
GRNa real time PCR forward：5'-ACCACATGGGGATGTTGC-3'；
GRNa real time PCR reverse：5'-CCAAGTCTCCGGCTGAAATA-3'。
(8) phenotype observation of zebra fish line with GRNa gene knockout
The obtained pure zebra fish with the GRNa knockout gene is bred, the body weight and the body length and other physiological standards are continuously measured in the culture process, the body weight and the body length of the zebra fish with the gene knockout gene are found to be lighter than those of wild zebra fish under the same breeding condition, the body length is shorter, and other pathological phenotypes are also found in an HE staining experiment, which is shown in an attached figure 5. These pathological phenotypes can be used for the study of the mechanism of GRNa in the treatment of neurological and metabolic diseases and related drug screening.
The zebra fish strain with the GRNa knockout obtained in the embodiment is proved to be capable of normally mating and spawning by more than three generations of mutual internal crossing, and capable of being stably inherited to offspring by the GRNa knockout. And confirmed at the genomic, mRNA level, confirming that GRNa gene is hardly expressed. Can be used as a tool for researching the zfGRNa gene. Can also be used for research of zebra fish science and application in the aspect of economic breeding, and has good commercial value.
Nucleotide or amino acid sequence listing
<110> university of Shanxi university
<120> preparation method of GRNa gene knockout zebra fish strain
<160>
<210>1
<211>19
<212> SgRNA1 site
<213>DNA
<400>1
TTTGTCCATTGCTGTCCTA
<210>2
<211>19
<212> SgRNA2 site
<213>DNA
<400>
TTATGTACCGAAGAGCCCT
<210>3
<211>19
<212> SgRNA3 site
<213>DNA
<400>
CTACTACTCCCAATGTGAT
<210>4
<211>18
<212> SgRNA4 site
<213>DNA
<400>
AAATGTGACGTAGCTGCG
<210>5
<211>18
<212> SgRNA5 site
<213>DNA
<400>
GTGCCCGTCCGTCCAATC
<210>6
<211>23
<212> primer Zebraphis GRNa sgRNA1 forward
<213>DNA
<400>
taggTTTGTCCATTGCTGTCCTA
<210>7
<211>23
<212> primer Zebraphis GRNa sgRNA1 reverse
<213>DNA
<400>
aaacTAGGACAGCAATGGACAAA
<210>8
<211>23
<212> primer Zebraphis GRNa sgRNA2 forward
<213>DNA
<400>
taggTTATGTACCGAAGAGCCCT
<210>9
<211>23
<212> primer Zebraphis GRNa sgRNA2 reverse
<213>DNA
<400>
aaacAGGGCTCTTCGGTACATAA
<210>10
<211>23
<212> primer Zebraphis GRNa sgRNA3 forward
<213>DNA
<400>
taggCTACTACTCCCAATGTGAT
<210>11
<211>23
<212> primer Zebraphis GRNa sgRNA3 reverse
<213>DNA
<400>
aaacATCACATTGGGAGTAGTAG
<210>12
<211>22
<212> primer Zebraphis GRNa sgRNA4 forward
<213>DNA
<400>
taggAAATGTGACGTAGCTGCG
<210>13
<211>23
<212> primer Zebraphis GRNa sgRNA4 reverse
<213>DNA
<400>
aaacCGCAGCTACGTCACATTTC
<210>14
<211>22
<212> primer Zebraphis GRNa sgRNA5 forward
<213>DNA
<400>
taggGTGCCCGTCCGTCCAATC
<210>15
<211>23
<212> primer Zebraphis GRNa sgRNA5 reverse
<213>DNA
<400>
aaacGATTGGACGGACGGGCACC
<210>16
<211>20
<212> primer grna exon15 TSF PCR nest forward
<213>DNA
<400>
AATCCCCCTGAGAAGGAAAA
<210>17
<211>20
<212> primer grna exon15 TSF PCR nest reverse
<213>DNA
<400>
CCAGGCCCAGTATTTGTGTT
<210>18
<211>21
<212> primer grna exon15 TSF PCR forward
<213>DNA
<400>
CCTGTCCTGATGGAAGCACAT
<210>19
<211>20
<212> primer grna exon15 TSF PCR reverse
<213>DNA
<400>
TGCGCATGCATTTTTAAGGC
<210>20
<211>18
<212> primer GRNa real time PCR forward
<213>DNA
<400>
5'-ACCACATGGGGATGTTGC-3'
<210>21
<211>20
<212> primer GRNa real time PCR reverse
<213>DNA
<400>
5'-CCAAGTCTCCGGCTGAAATA-3'
Claims (3)
1. A preparation method of a GRNa gene knockout zebra fish strain is characterized by comprising the following steps:
(1) sgRNA for screening targeted GRNa gene
Searching a genome sequence of a zebra fish GRNa gene from NCBI, selecting five sgRNA binding sites in an exon region of a coded protein by utilizing sgRNA design software, designing corresponding sgRNA primers, annealing the sgRNA primers, and connecting the sgRNA primers to a sgRNA expression vector;
(2) preparation of sgRNA and Cas9mRNA of zebra fish GRNa gene
Obtaining sgRNA and Cas9mRNA of the GRNa gene of the zebra fish by in vitro transcription by using an in vitro transcription kit;
(3) microinjection of sgRNA and Cas9mRNA into zebra fish fertilized egg single cell stage
The male and female zebra fish are respectively placed into a mating tank at night before microinjection and separated by a baffle plate, the baffle plate is pulled out in the morning next day, the male and female zebra fish are mated, fertilized eggs are collected, and Cas9mRNA and sgRNA mRNA of a target gene GRNa are injected into the fertilized eggs under a microscope within half an hour;
(4) target efficiency detection
After microinjection is carried out for 24 hours, each sgRNA and Cas9 injection embryo 4 tubes are collected, five eggs in each tube are extracted, genome DNA is extracted, PCR amplification is carried out on a targeting region, and the sgRNA with the highest cutting activity is screened by a T7E1 method;
(5) culturing the embryo with the targeting effect to adult fish, and performing internal crossing to lay eggs; randomly taking tube embryos, taking 5 eggs per tube, extracting a genome, carrying out PCR amplification on a targeting area, and carrying out enzyme digestion on T7E1 to identify whether targeting exists;
(6) culturing embryos with the targeting effect to adult fishes, cutting tails one by one to extract genomes, carrying out PCR amplification on targeting areas, sending the amplified target areas to a company for sequencing to see whether base insertion or deletion exists and frame shift mutation or nonsense mutation is caused, and thus effective targeting is achieved;
(7) breeding the heterozygote or homozygote zebra fish which are effectively targeted to adult fish, and mating and spawning to obtain the next generation of zebra fish; determining the F0 generation of the zebra fish mutant by screening, respectively hybridizing the F0 generation mutant with wild zebra fish to obtain F1 generation embryos, culturing at 28 ℃, and observing the survival rate of the F1 generation embryos at the initial stage; after fertilization for two days, 5 embryos are respectively taken from each mutant F1 generation for mutation inheritance identification; extracting genome of each embryo separately, PCR amplifying the region near the target site, sending to company for sequencing and identification to determine whether the mutation can be inherited to F1 generation;
(8) breeding the zebrafish mutant in the F1 generation to adult fish if the presence of the sense mutation is detected from the F1 generation embryo; respectively carrying out tail shearing on each F1 generation adult zebra fish, and screening F1 generation mutants;
(9) hybridizing female fishes and male fishes in the F1 generation mutant to obtain F2 generation embryos, culturing the F2 generation embryos at 28 ℃, observing the development condition of the embryos, and knocking out genes to prevent the embryos from dying if the embryos are normally developed; culturing F2 embryo to adult fish, cutting tail, extracting genome, PCR sequencing, and identifying genotype to see whether homozygote exists; carrying out subsequent experimental study on heterozygote or homozygote zebra fish after the genotype identification;
(10) and hybridizing the F2 generation mutant homozygote adult fish female fish and male fish to obtain F3 generation, namely the GRNa gene knockout zebra fish strain.
2. The method of claim 1, wherein the five sgRNA sites are each:
SgRNA1：TTTGTCCATTGCTGTCCTA；
SgRNA2：TTATGTACCGAAGAGCCCT；
SgRNA3：CTACTACTCCCAATGTGAT；
SgRNA4：AAATGTGACGTAGCTGCG；
SgRNA5：GTGCCCGTCCGTCCAATC。
3. the method of claim 1 or 2, wherein the GRNa knockout zebra fish line is used for researching the mechanism of GRNa in the treatment of nervous system diseases and metabolic diseases and the like and the screening of related drugs.
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