Method for creating hemorrhagic disease-resistant grass carp by knocking out dhcr7 gene and application of hemorrhagic disease-resistant grass carp

Abstract

The invention belongs to the technical field of aquatic organism breeding, and discloses a method for creating anti-hemorrhagic disease grass carp by knocking out a dhcr7 gene and application of the anti-hemorrhagic disease grass carp, insertion / deletion mutation is generated in the grass carp dhcr7 gene by utilizing a CRISPR-Cas9 technology, so that the dhcr7 gene cannot encode protein or the encoded protein does not have functions. According to the invention, the innate immune negative regulation gene dhcr7 is knocked out of the grass carp for the first time, an F0 generation group with a high mutation rate is obtained, dhcr7 knockout is found to significantly improve the resistance of the grass carp to GCRV-II virus, a method and a breeding material are provided for breeding the hemorrhagic disease-resistant grass carp, and the method has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of aquatic organism breeding technology, and particularly relates to a method of breeding by knocking out... dhcr7 Methods for genetically creating grass carp resistant to hemorrhagic disease and their applications. Background Technology

[0002] grass carp( Ctenopharyngodon idella Grass carp (Ctenopharynx spp.) belongs to the class Osteichthyes, order Cypriniformes, family Cyprinidae, subfamily Leuciscinae, and genus Ctenopharynx. It is a fast-growing, highly adaptable, relatively large freshwater fish with flesh that appeals to a wide range of palates. According to the *China Fisheries Statistical Yearbook: 2025*, my country's grass carp farming output reached 6.1648 million tons in 2024, accounting for approximately 17.4% of total freshwater aquaculture output, ranking first among all fish species. Grass carp farming provides a large amount of high-quality food protein, making a significant contribution to improving residents' diets and ensuring their health, and thus possessing significant economic value.

[0003] Grass carp hemorrhagic disease is an acute, fatal infectious disease caused by grass carp reovirus (GCRV). It is widespread in major grass carp farming areas in my country, typically breaking out from June to September, primarily affecting one-year-old grass carp. It is estimated that the economic losses caused by grass carp hemorrhagic disease in my country exceed 1 billion yuan annually. GCRV belongs to the Reoviridae family and the Aquatic Reovirus genus. It is a non-enveloped dsRNA virus composed of 11 double-stranded RNA segments and two capsid proteins. Sequence analysis shows that GCRV can be divided into three subtypes: GCRV-I, GCRV-II, and GCRV-III. Among them, GCRV-II is the predominantly circulating strain in my country, exhibiting the strongest virulence. Infection can cause lesions in multiple organs and tissues, including the kidneys and liver, leading to severe hemorrhagic symptoms and a mortality rate as high as 80%. Currently, the infection and pathogenesis mechanisms of GCRV-II are unclear, and there are no highly effective treatments.

[0004] The innate immune response is the first line of defense against viruses in fish. Viral invasion activates a series of innate immune responses through different signal transduction pathways, such as the interferon pathway. Dhcr7 (7-dehydrocholesterol reductase), the final catalytic enzyme in the cholesterol synthesis pathway, has been shown to negatively regulate the innate immune response in mice and humans. Our previous studies have also confirmed this in grass carp. dhcr7 It is a gene that negatively regulates innate immunity. This invention utilizes CRISPR-Cas9 technology to knock it out in grass carp. dhcr7 This invention enhances the innate immune response of grass carp to GCRV-II infection, thereby achieving resistance to hemorrhagic disease. The novel hemorrhagic disease-resistant grass carp germplasm created in this invention will reduce economic losses caused by disease in grass carp farming and decrease the amount of veterinary drugs used, possessing significant industrial application value. Summary of the Invention

[0005] The purpose of this invention is to provide a method for knocking out dhcr7 Methods and applications of genetically engineered grass carp resistant to hemorrhagic disease, which utilize CRISPR-Cas9 technology in grass carp... dhcr7 Introducing insertion / deletion mutations into genes disrupts their function. This invention confirms... dhcr7 Gene knockout grass carp is an effective new germplasm resistant to hemorrhagic disease, providing a method and example for the breeding of new grass carp varieties resistant to hemorrhagic disease. It is expected to reduce economic losses caused by disease in grass carp farming and has important industrial application value.

[0006] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a method for knocking out dhcr7 A method for genetically creating grass carp resistant to hemorrhagic disease involves using CRISPR-Cas9 technology in grass carp. dhcr7 Insertion / deletion mutations occur in genes, making dhcr7 Genes that cannot encode proteins or whose encoded proteins are not functional.

[0007] The above technical solution includes the following steps: Step 1: Combine Cas9 protein and dhcr7 The sgRNA was mixed and incubated at room temperature to form the RNP complex, which was then microinjected into grass carp 1-cell stage fertilized eggs. Step 2: Detect the mutation rate of the grass carp injected in Step 1, and screen for those with a mutation rate between 50% and 100%. dhcr7 Knock out the F0 generation grass carp population.

[0008] In the above technical solution, in step 1, the Cas9 protein and dhcr7 The molar ratio of sgRNA was 1:2, the concentration of Cas9 protein was 300 ng / μl, and the concentration of sgRNA was 150 ng / μl.

[0009] In the above technical solution, in step 1, the target sequence of sgRNA is as shown in SEQ ID NO. 2, which is 5'-GGAAAGGAGCCGGCACAGTG-3'.

[0010] In the above technical solution, in step 1, the microinjection method is as follows: wrap the containers containing grass carp eggs and semen with tin foil and a towel respectively, and store them on ice; every 15 minutes, take out a portion of eggs and semen for dry artificial insemination, and then perform microinjection; stop the injection after 15 minutes, and repeat artificial insemination until the grass carp eggs have been stored on ice for more than 90 minutes; use a micrometer to measure the size of the injection droplets to ensure that the injection volume of each fertilized egg is 1-2 nl.

[0011] In the above technical solution, in step 2, the method for detecting the mutation rate is PAGE electrophoresis, TA cloning and Sanger sequencing.

[0012] In the above technical solution, the specific method is as follows: the injected grass carp embryos are cultured to 1-2 months old, the tail fin is cut to extract genomic DNA, a DNA fragment containing the sgRNA target point is amplified by PCR, and chimeric mutant individuals are screened by PAGE electrophoresis; individuals with representative band patterns are selected for Sanger sequencing to further confirm the existence of mutations and evaluate the mutation rate; chimeric individuals with a mutation rate of 50-100% are selected, and the specific mutation type is identified by TA cloning and Sanger sequencing. dhcr7 dhcr7

[0013] In a second aspect, the application provides the above method for creating grass carp resistant to hemorrhagic disease.

[0014] In a third aspect, the application provides an sgRNA, the target sequence of which is shown in SEQ ID NO. 2, which is 5'-GGAAAGGAGCCGGCACAGTG-3'.

[0015] In a fourth aspect, the application provides the above sgRNA for creating grass carp resistant to hemorrhagic disease.

[0016] The application has the beneficial effects that the application first knocks out the innate immune negative regulation gene dhcr7 in grass carp, obtains a high mutation rate F0 population, and finds that dhcr7 knockout significantly improves the resistance of grass carp to GCRV-II virus, provides a method and breeding material for breeding grass carp resistant to hemorrhagic disease, and has important industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The sgRNA target point design and high-efficiency sgRNA screening results for grass carp dhcr7

[0018] Figure 2 The mutation efficiency detection and mutation type identification results for gene-edited grass carp dhcr7

[0019] Figure 3 The GCRV-II challenge experiment results of the high mutation rate F0 generation of grass carp, which shows the survival rate and hemorrhagic disease symptoms of the mutant grass carp after GCRV-II virus infection. dhcr7 dhcr7 dhcr7 dhcr7 The high mutation rate F0 generation of grass carp (the same below).​​​​​​​

[0020] Figure 4 For dhcr7 The liver tissue pathological changes of high mutation rate F0 generation grass carp after GCRV-II infection, GCRV-II infection dhcr7 The liver lesion degree of mutant grass carp is lighter.

[0021] Figure 5 For dhcr7 The expression situation of innate immune antiviral genes in liver, spleen, intestinal tract and kidney tissues of high mutation rate F0 generation grass carp after GCRV-II infection IFN1 , dhcr7 The expression in liver, spleen, intestine and kidney of mutant grass carp IFN1 is significantly higher than that of wild type.

[0022] Figure 6 For dhcr7 The expression situation of innate immune antiviral genes in liver, spleen, intestinal tract and kidney tissues of high mutation rate F0 generation grass carp after GCRV-II infection IFI56 , dhcr7 The expression in liver, spleen, intestine and kidney of mutant grass carp IFI56 is significantly higher than that of wild type. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples. The present application can be implemented in many different forms, and should not be understood as being limited to the examples set forth herein. On the contrary, these examples are provided so that the present disclosure will be thorough and complete, and will fully convey the idea of the present application to those skilled in the art, and the present application will be limited only by the claims.

[0024] Unless specifically stated, the technical means adopted by the present application are all conventional schemes in the art; the reagents and materials are all from commercial channels.

[0025] The present application is directed to grass carp dhcr7 Four sgRNAs are designed and synthesized. Preferably, one high-efficiency sgRNA is obtained by in vitro cleavage experiment, and the targeting sequence is 5'-GGAAAGGAGCCGGCACAGTG-3'(SEQ ID NO.2). The Cas9 protein and the sgRNA targeting grass carp dhcr7 Gene are mixed at a molar ratio of 1:2, incubated at room temperature to form an RNP complex, wherein the concentration of Cas9 protein is 300 ng / μl, and the concentration of sgRNA is 150 ng / μl, which is used for microinjection of grass carp fertilized eggs.

[0026] The Cas9 protein and dhcr7The RNP formed by sgRNA incubation is microinjected into artificially bred grass carp zygotes, and the injection volume is 1-2 nl. Preferably, the container containing grass carp eggs and sperm is wrapped with tin foil and a towel respectively, and stored on ice. Every 15 minutes, part of the eggs and sperm are taken out for dry artificial insemination, and then microinjection is performed. If more than 15 minutes, stop injection, re-inseminate, until the grass carp eggs are stored on ice for more than 90 minutes. The size of the injection droplet is measured using a micrometer to ensure that the injection volume of each zygote is 1-2 nl. The above microinjection scheme has little damage to the grass carp zygote, and the hatching rate and gene editing efficiency are higher.

[0027] The mutation rate and mutation type of the injected grass carp are detected by PAGE electrophoresis, TA cloning and Sanger sequencing, and a dhcr7 high mutation rate (50-100%) F0 generation grass carp population is obtained. Preferably, the injected grass carp embryos are cultured to 1-2 months old, the tail fin is cut to extract genomic DNA, the DNA fragment containing the dhcr7 sgRNA target point is amplified by PCR, and the chimeric mutant individuals are screened by PAGE electrophoresis. Some individuals with representative band patterns are selected for Sanger sequencing to further confirm the existence of mutations and evaluate the mutation rate. Several high mutation rate chimeric individuals are selected, and the specific dhcr7 mutation type is identified by TA cloning and Sanger sequencing.

[0028] The dhcr7 high mutation rate F0 generation grass carp population constructed by the above method is cultured to 5-6 months old, and a GCRV-II virus challenge experiment is performed to evaluate its ability to resist hemorrhagic disease. Preferably, wild-type grass carp with a body length of 8-10 cm and dhcr7 high mutation rate F0 generation grass carp are selected as GCRV-II challenge objects. The soaking method is used for challenge, and the symptoms of grass carp hemorrhagic disease and the death time of grass carp are observed, and the challenge is continued until the death of the challenged grass carp is stable. The survival rate statistics, histopathological analysis and innate immune gene expression detection are used to systematically evaluate dhcr7 the knockout grass carp's ability to resist hemorrhagic disease. The results show that, dhcr7 the survival rate of the high mutation rate F0 generation grass carp is higher than that of the wild-type grass carp by about 40%, the liver lesion degree is lighter and the recovery time is shorter, and the expression of innate immune genes in liver, spleen, intestine and kidney is significantly enhanced, confirming IFN1 knockout grass carp is an effective new germplasm for anti-hemorrhagic disease grass carp. IFI56 dhcr7

[0029] Example 1: Grass carp dhcr7 sgRNA design, synthesis and screening 1.1 Grass carp dhcr7 sgRNA target point design​​ According to grass carp dhcr7 Sequence information designed 4 sgRNA target points. In this embodiment, the grass carp dhcr7 Gene sequence was downloaded from NCBI database, and the corresponding transcript sequence number was XM_051900158.1. The grass carp dhcr7 The gene contains 8 exons. In this embodiment, 2 sgRNA target points were designed in the 2nd and 3rd exons respectively (Table 1). Figure 1 A), and the sequences are shown in Table 1.

[0030] Table 1 sgRNA target points of grass carp dhcr7 Gene sgRNA 1.2 sgRNA in vitro transcription and purification of grass carp dhcr7 Gene PCR was used to amplify the DNA template for in vitro transcription of sgRNA. The primers used were sgRNA-specific forward primers and universal reverse primers, and the sequences are shown in Table 2. The PCR reagent used was PowerPol 2X PCR Mix (purchased from Abclonal, product number RK20718), and the reaction system was prepared according to the instructions, and sgRNA-specific forward primers and universal reverse primers and sgRNA backbone templates were added. The PCR reaction program was: 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 68°C annealing and extension for 30 s, 35 cycles; 72°C final extension for 5 min. After the amplification was completed, the PCR product was subjected to agarose gel electrophoresis, the gel concentration was 2%, and the band size was about 132 bp without obvious impurity band, and the target band was recovered by cutting the gel. According to the instructions of T7 High Yield RNA Transcription Kit (purchased from Vazyme, product number TR101), the sgRNA of grass carp dhcr7In vitro transcription of sgRNA was performed using a 20 μl reaction system, with 1 μg of DNA template added. The mixture was incubated at 37°C for 4 hours, followed by the addition of 1 μl of DNase I and incubation at 37°C for another 15 minutes to remove the DNA template. The transcription efficiency was verified by agarose gel electrophoresis. The transcribed sgRNA was purified and recovered using lithium chloride precipitation. The specific steps were as follows: 30 μl of 7.5 M lithium chloride solution and 30 μl of RNase-free water were added to the 20 μl in vitro transcription reaction system, mixed, and incubated overnight at -20°C. The mixture was centrifuged at 12000g for 15 minutes at 4°C, the supernatant was discarded, and the precipitate was washed once with 500 μl of pre-chilled 75% anhydrous ethanol. The precipitate was then centrifuged at 12000g for 5 minutes at 4°C, the supernatant was discarded, and the precipitate was allowed to air dry for 5 minutes. 20 μl of RNase-free water was added to dissolve the RNA precipitate. Take 1 μL of sgRNA solution, detect its purity and concentration using a micro spectrophotometer, aliquot it, and store it at -80℃ for later use.

[0031] Table 2 Grass carp dhcr7 Gene sgRNA in vitro transcription template PCR amplification primers 1.3 Grass carp dhcr7 Validation of gene sgRNA cleavage efficiency Grass carp containing the above-mentioned sgRNA target were amplified by PCR. dhcr7 Gene fragments were used for in vitro Cas9 RNP cleavage experiments. A 20 μl reaction mixture was prepared, containing 500 ng of Cas9 protein (purchased from Novoprotein, catalog number E365), 250 ng of sgRNA, 2 μl of 10×Reaction Buffer, and 200 ng of dsDNA substrate. The cleavage reaction was completed by incubation at 37°C for 1 hour, followed by incubation at 95°C for 5 minutes to denature the Cas9-sgRNA RNP complex. The sgRNA cleavage efficiency was then assessed by agarose gel electrophoresis (gel concentration 1.5%). Figure 1 As shown in B, grass carp dhcr7 The cleavage efficiency of gene 2 sgRNA is the highest.

[0032] Table 3 Grass carp dhcr7 Primers for amplifying gene sgRNA target fragments Example 2: dhcr7 Gene knockout grass carp creation 2.1 Grass carp embryo microinjection Grass carp fertilized eggs were obtained through artificial spawning induction and artificial insemination, and the Cas9 protein was then combined with... dhcr7The RNP formed by incubation of gene 2 sgRNA (Cas9 protein working concentration of 300 ng / μl, sgRNA working concentration of 150 ng / μl) was microinjected into 1-cell stage grass carp zygotes. The specific steps were as follows: 50 ml centrifuge tubes containing grass carp eggs and semen were wrapped separately with aluminum foil and a towel and stored on ice. Every 15 minutes, a portion of eggs and semen was collected and dry artificial insemination was performed in a 90 mm culture dish. The artificially inseminated grass carp embryos were arranged in the 90 mm culture dish, excess water was removed, and microinjection was performed. The size of the injection droplet was measured using a micrometer to ensure a droplet diameter of 120-150 μm, corresponding to an injection volume of 1-2 nl. The injection window for each batch of grass carp zygotes was approximately 15 minutes; if this window was exceeded, injection was stopped, and artificial insemination was repeated. Unfertilized grass carp eggs were stored on ice for no more than 90 minutes. After injection, the grass carp fertilized eggs were placed in incubation tanks for hatching, with the water temperature controlled at 23-28℃ and sufficient dissolved oxygen ensured. Using the above protocol, four microinjections of grass carp embryos were performed in May 2024, injecting approximately 8,000 grass carp fertilized eggs. On the 5th day after injection, approximately 1,000 F0 generation fish were collected. dhcr7 Remove grass carp fry.

[0033] 2.2 Grass carp dhcr7 Mutation identification and screening of high mutation rate F0 generation chimeric individuals Successfully hatched grass carp embryos were cultured to 1-2 months of age, and genomic DNA was extracted from the tail fins. This DNA was then amplified by PCR. dhcr7 The DNA fragment targeting gene 2 sgRNA was selected, and the primer sequences used are shown in Table 3. The PCR products were subjected to PAGE electrophoresis, and several individuals with electrophoretic bands significantly different from the wild-type control were selected for sequencing verification. Figure 2 As shown in Figure A, [the substance] was detected in the injected grass carp fry. dhcr7 High-frequency gene mutations. For F0 generation individuals with high mutation rates confirmed by Sanger sequencing, their PCR products were excised from the gel, ligated into the pMD18-T vector (purchased from Takara, catalog number 6011), single clones were picked and Sanger sequencing was performed again to identify specific mutations. dhcr7 Mutation type. For example... Figure 2 As shown in B, a total of 19 species of grass carp were identified. dhcr7 The types of gene mutations were identified, and the proportion of each mutation was statistically analyzed. PAGE electrophoresis was used to analyze the mutation types. dhcr7 Further mutation screening was conducted on F0 generation grass carp individuals, and chimeric individuals with a mutation rate of over 50% were selected as... dhcr7 The high-mutation-rate F0 generation population continued to be cultured. Using the above method, approximately 500 grass carp were obtained from the F0 generation produced by injection in 2024. dhcr7 Individuals with high mutation rates.

[0034] Example 3: dhcr7 Gene knockout grass carp creation 3.1 Grass carp embryo microinjection Grass carp fertilized eggs were obtained through artificial spawning induction and artificial insemination, and the Cas9 protein was then combined with... dhcr7 The RNP formed by incubation with gene 2 sgRNA (Cas9 protein working concentration of 300 ng / μl, sgRNA working concentration of 150 ng / μl) was microinjected into 1-cell stage grass carp zygotes. The specific steps are as described above. Using the above protocol, two grass carp embryo microinjection trials were performed in May 2025, injecting approximately 6000 grass carp zygotes. On day 5 post-injection, approximately 200 F0 generation embryos were collected. dhcr7 Remove grass carp fry.

[0035] 3.2 Grass carp dhcr7 Mutation identification and screening of high mutation rate F0 generation chimeric individuals Successfully hatched grass carp embryos were cultured to 1-2 months of age, and genomic DNA was extracted from the tail fins. This DNA was then amplified by PCR. dhcr7 The DNA fragment targeting gene 2 sgRNA was analyzed, and the primer sequences used are shown in Table 3. PAGE electrophoresis was performed on the PCR products, and individuals with significantly different electrophoretic bands from the wild-type control were screened. Several chimeric individuals were selected for Sanger sequencing verification. Chimeric individuals with a mutation rate of over 50% were selected as... dhcr7 The high-mutation-rate F0 generation population continued to be cultured. Using the above method, 110 grass carp were obtained from the F0 generation produced by injection in 2025. dhcr7 Individuals with high mutation rates accounted for 85% of the total.

[0036] Example 4: F0 generation dhcr7 The survival rate of knocked-out grass carp after viral infection was significantly higher than that of wild-type grass carp. The obtained F0 generation dhcr7 Grass carp with high mutation rates are raised to 5-6 months of age, selecting wild-type grass carp with a body length of 8-10cm and F0 generation. dhcr7 Grass carp with a high mutation rate were challenged with GCRV-II. The immersion method was used for challenge, and the symptoms of hemorrhagic disease and the time of death were observed and recorded until the mortality rate of the challenged grass carp stabilized. The specific steps were as follows: A temporary holding and challenge device for grass carp was set up indoors, ensuring sufficient dissolved oxygen and a water temperature above 28℃. The virus stock solution was removed from a -80℃ freezer and placed in 28℃ water for 2 hours for resuscitation, then diluted 1:800 in 60L of aerated water. 75 wild-type control grass carp and 75 wild-type control grass carp were used. dhcr7Grass carp with a high mutation rate were infected by immersing them in 30L of viral working solution for 30 minutes. After GCRV-II infection, the grass carp were transferred to six pre-prepared rearing tanks, each filled with approximately 50L of water and stocked with 25 fish. Feeding frequency was reduced during the challenge period, and the disease status and mortality of the grass carp were observed daily. The experiment was stopped 21 days after challenge, and the numbers of wild-type and... dhcr7 Survival rate of grass carp with high mutation rates. For example... Figure 3 As shown, at the end of the challenge experiment, dhcr7 The survival rate of grass carp with a high mutation rate is about 40% higher than that of wild-type grass carp, indicating that... dhcr7 Knockout grass carp showed a significantly enhanced ability to resist GCRV-II virus infection.

[0037] Example 5: F0 generation dhcr7 The liver lesions in grass carp were relatively mild during the viral challenge process. Wild-type grass carp with a body length of 8-10cm and F0 generation were respectively tested. dhcr7 Grass carp with a high mutation rate were challenged with GCRV-II. The immersion method was used for challenge, with the specific steps described above. Samples were taken at the following time points after viral infection: D0 (the day of the challenge experiment), D1, D3, D5, D7, D9, D11, and D13. At each time point, several wild-type and... dhcr7 Grass carp with a high mutation rate were photographed to record their appearance. Liver tissue was obtained from dissected fish, fixed overnight in Born's solution, and then embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and scanned on slides to observe liver lesions during the challenge process. Figure 4 As shown, dhcr7 The knockout grass carp liver tissue showed milder vacuolation, and by day 9 post-infection, it had largely returned to normal; while the wild-type grass carp liver tissue exhibited significant vacuolation, with apoptosis observed, indicating that... dhcr7 Knockout grass carp showed a significantly enhanced ability to resist GCRV-II virus infection.

[0038] Example 6: F0 generation dhcr7 Knockout grass carp showed a significantly enhanced innate immune response during the challenge process. Wild-type grass carp with a body length of 8-10cm and F0 generation were respectively tested. dhcr7 Grass carp with a high mutation rate were challenged with GCRV-II. The immersion method was used for challenge, and the specific steps are as described above. Samples were taken at the following time points after viral infection: D0 (the day of the challenge experiment), D1, D3, D5, D7, D9, and D11. At each time point, several wild-type and... dhcr7High mutation rate grass carp, dissect the fish body, take the liver, spleen, intestinal and kidney tissues in 1.5 ml centrifuge tube, use Trizol method to extract RNA, -80℃ refrigerator preservation. According to the reverse transcription kit (purchased from Vazyme, goods number R312) instruction carries out reverse, obtains cDNA first chain, -20℃ refrigerator preservation. According to SYBR Green qPCR Mix (purchased from Vazyme, goods number Q712) instruction carries out real-time fluorescent quantitative PCR experiment, detects the expression level of grass carp innate immune gene IFN1 and IFI56 . As shown in Figure 5 and Figure 6 , dhcr7 The expression level of grass carp in liver, spleen, intestine and kidney IFN1 and IFI56 is significantly higher than that of wild type grass carp from the 3rd to the 7th day after challenge, which shows that dhcr7 knockout can activate grass carp innate immune antiviral response to a greater extent when infected with GCRV-II, thereby exerting the effect of anti-hemorrhagic disease.

[0039] Obviously, the above examples are only examples for clearly illustrating, but not limit the embodiments. For those skilled in the art, on the basis of the above description, other different forms of changes or variations can also be made. Here, it is not necessary and also cannot be exhaustive for all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method of creating an anti-hemorrhagic grass carp by knocking out dhcr7 a gene characterized in that: The method is to produce insertion / deletion mutation in grass carp dhcr7 gene by using CRISPR-Cas9 technology, so that dhcr7 the gene cannot encode protein or the encoded protein does not have function.

2. The method of claim 1, wherein: comprising the following steps: Step 1: Mix Cas9 protein and sgRNA, incubate at room temperature to form RNP complex, microinject into grass carp 1-cell stage fertilized eggs; dhcr7 Step 1: Mix Cas9 protein and sgRNA, incubate at room temperature to form RNP complex, microinject into grass carp 1-cell stage fertilized eggs; Step 2: Detect the mutation rate of grass carp injected in step 1, and screen to obtain the grass carp with mutation rate of 50-100% dhcr7 Knockout F0 generation of grass carp population.

3. The method of claim 2, wherein: In step 1, the Cas9 protein and dhcr7 The molar ratio of sgRNA was 1:2, the concentration of Cas9 protein was 300 ng / μl, and the concentration of sgRNA was 150 ng / μl.

4. The method of claim 2, wherein: In step 1, the target sequence of sgRNA is 5'-GGAAAGGAGCCGGCACAGTG-3' as shown in SEQ ID NO.

2.

5. The method of claim 2, wherein: In step 1, the method of microinjection is as follows: the container containing grass carp eggs and sperm is wrapped with tin foil and towel respectively, and stored on ice; every 15 minutes, part of the eggs and sperm are taken out for dry artificial insemination, and then microinjection is performed; if more than 15 minutes, stop injection, re-perform artificial insemination, until the time of storing grass carp eggs on ice is more than 90 minutes; use micrometer to measure the size of injection droplet, and ensure that the injection volume of each fertilized egg is 1-2 nl.

6. The method of claim 2, wherein: In step 2, the method for detecting mutation rate is PAGE electrophoresis, TA cloning and Sanger sequencing.

7. The method of claim 6, wherein: The specific method is as follows: Injected grass carp embryos are cultured to 1-2 months of age, genomic DNA is extracted from the tail fin, and PCR amplification is performed on the embryos containing... dhcr7 DNA fragments targeting sgRNA were screened for chimeric mutants using PAGE electrophoresis; individuals with representative banding patterns were selected for Sanger sequencing to further confirm the presence of mutations and assess the mutation rate; chimeric individuals with mutation rates between 50-100% were selected for specific identification using TA cloning and Sanger sequencing. dhcr7 Mutation type.

8. The method of any one of claims 1-7 for use in creating grass carp resistant to hemorrhagic disease.

9. An sgRNA, characterized in that: The target sequence thereof is 5'-GGAAAGGAGCCGGCACAGTG-3' as shown in SEQ ID NO.

2.

10. The sgRNA of claim 9 for use in creating grass carp resistant to hemorrhagic disease.

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