Method for creating anti-hemorrhagic grass carp by knocking out dhcr7 gene and application thereof

Abstract

The application belongs to the technical field of aquatic organism breeding, and discloses a method for creating grass carp resistant to hemorrhagic disease by knocking out dhcr7 a gene and application thereof. dhcr7 The method utilizes CRISPR-Cas9 technology to generate an insertion / deletion mutation in a grass carp dhcr7 gene, so that the gene cannot encode a protein or the encoded protein does not have a function. dhcr7 The application first knocks out the innate immune negative regulation gene dhcr7 in grass carp, obtains a F0 generation population with a high mutation rate, and finds that knocking out significantly improves the resistance of grass carp to GCRV-II virus, thereby providing a method and breeding material for breeding grass carp resistant to hemorrhagic disease and having 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:

[0007] 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.

[0008] The above technical solution includes the following steps:

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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'.

[0013] 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.

[0014] In the above technical solutions, step 2 involves detecting the mutation rate using PAGE electrophoresis, TA cloning, and Sanger sequencing.

[0015] In the above technical solution, the specific method is as follows: the 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.

[0016] Secondly, the present invention provides the application of the above-mentioned method in the creation of grass carp resistant to hemorrhagic disease.

[0017] Thirdly, the present invention provides an sgRNA whose target sequence is shown in SEQ ID NO. 2, which is 5'-GGAAAGGAGCCGGCACAGTG-3'.

[0018] Fourthly, this invention provides the application of the above-mentioned sgRNA in the creation of grass carp resistant to hemorrhagic disease.

[0019] The beneficial effects of this invention are as follows: This invention is the first to knock out the gene that negatively regulates innate immunity in grass carp. dhcr7 A high mutation rate F0 generation population was obtained, and it was discovered that... dhcr7 Knockout significantly enhances grass carp's resistance to GCRV-II virus, providing a method and breeding material for breeding grass carp resistant to hemorrhagic disease, and has important industrial application value. Attached Figure Description

[0020] Figure 1 grass carp dhcr7 Gene sgRNA target design and efficient sgRNA screening results.

[0021] Figure 2 for dhcr7 Results of gene-edited grass carp mutation efficiency detection and mutation type identification.

[0022] Figure 3 for dhcr7 Results of GCRV-II challenge experiments on high mutation rate F0 generation grass carp populations demonstrate dhcr7 Survival rate and hemorrhagic septicemia symptoms in mutant grass carp after GCRV-II virus infection. WT indicates wild-type grass carp. dhcr7 express dhcr7 High mutation rate F0 generation grass carp (the same applies below).

[0023] Figure 4 for dhcr7 Pathological changes in liver tissue of F0 generation grass carp with high mutation rate after GCRV-II infection; GCRV-II infection dhcr7 The mutant grass carp had milder liver lesions.

[0024] Figure 5 for dhcr7 High mutation rate F0 generation grass carp showed innate immune antiviral genes in liver, spleen, intestine and kidney tissues after GCRV-II infection. IFN1 The expression situation, dhcr Mutant grass carp liver, spleen, intestines, and kidneys ​ Expression was significantly higher than that of the wild type.

[0025] ​ for ​ High mutation rate F0 generation grass carp showed innate immune antiviral genes in liver, spleen, intestine and kidney tissues after GCRV-II infection. ​ The expression situation, ​ Mutant grass carp liver, spleen, intestines, and kidneys ​ Expression was significantly higher than that of the wild type. Detailed Implementation

[0026] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0027] Unless otherwise specified, the technical means used in this invention are all conventional solutions in the field; the reagents and materials are all from commercial sources.

[0028] This invention targets grass carp ​Four sgRNAs were designed and synthesized. Preferably, one highly efficient sgRNA was obtained through in vitro cleavage experiments, with its targeting sequence being 5'-GGAAAGGAGCCGGCACAGTG-3' (SEQ ID NO.2). The Cas9 protein was then used to target grass carp. ​ The sgRNA of the gene was mixed at a 1:2 molar ratio and incubated at room temperature to form the RNP complex, in which the concentration of Cas9 protein was 300 ng / μl and the concentration of sgRNA was 150 ng / μl, and was used for microinjection into grass carp fertilized eggs.

[0029] Cas9 protein and ​ The RNP formed by sgRNA incubation was microinjected into artificially bred grass carp zygotes at a volume of 1-2 nmol. Preferably, the containers containing grass carp eggs and semen were wrapped separately with aluminum foil and a towel, and stored on ice. Every 15 minutes, portions of eggs and semen were removed for dry insemination, followed by microinjection. Injection was stopped after 15 minutes, and artificial insemination was repeated until the grass carp eggs had been stored on ice for more than 90 minutes. The size of the injection droplet was measured using a micrometer to ensure that the injection volume for each zygote was 1-2 nmol. This microinjection protocol causes minimal damage to grass carp zygotes and results in higher hatching rates and gene editing efficiency.

[0030] The mutation rate and mutation type of injected grass carp were detected by PAGE electrophoresis, TA cloning, and Sanger sequencing, and selected the appropriate mutations. ​ A high mutation rate (50-100%) F0 generation grass carp population. Preferably, the injected grass carp embryos are cultured to 1-2 months of age, and genomic DNA is extracted from the tail fins and amplified by PCR. ​ DNA fragments targeting sgRNA were screened for chimeric mutants using PAGE electrophoresis. Individuals exhibiting representative banding patterns were selected for Sanger sequencing to further confirm mutation presence and assess mutation rate. Several chimeric individuals with high mutation rates were selected for identification using TA cloning and Sanger sequencing. ​ Mutation type.

[0031] The structure constructed by the above method ​ High-mutation-rate F0 generation grass carp were cultured to 5-6 months of age and then challenged with GCRV-II virus to evaluate their resistance to hemorrhagic disease. Preferably, wild-type grass carp with a body length of 8-10 cm were selected. ​ High-mutation-rate F0 generation grass carp were used as subjects for GCRV-II challenge. 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 of the challenged grass carp stabilized. Survival rate statistics, histopathological analysis, and innate immune gene expression detection were used to systematically evaluate... ​ The ability of grass carp to resist hemorrhagic disease was knocked out. The results showed that... ​The F0 generation grass carp with a high mutation rate had a survival rate of approximately 40% higher than wild-type grass carp after viral challenge. Their liver lesions were less severe and their recovery time was shorter. Furthermore, they exhibited higher levels of innate immune genes in their liver, spleen, intestines, and kidneys. ​ and ​ The expression was significantly enhanced, confirming ​ Knockout grass carp is an effective new germplasm for grass carp resistant to hemorrhagic disease.

[0032] Example 1: Grass carp ​ Gene sgRNA design, synthesis and screening

[0033] 1.1 Grass carp ​ Gene sgRNA target design

[0034] According to grass carp ​ Four sgRNA targets were designed based on the sequence information. In this example, grass carp... ​ The gene sequence was downloaded from the NCBI database, and the corresponding transcript sequence number is XM_051900158.1. Grass carp ​ The gene contains 8 exons. In this example, two sgRNA targets were designed in each of its exons 2 and 3. ​ A), whose sequence is shown in Table 1.

[0035] Table 1 Grass carp ​ Gene sgRNA target

[0036]

[0037] 1.2 Grass carp ​ In vitro transcription and purification of gene sgRNA

[0038] DNA templates for in vitro transcription of sgRNA were obtained by PCR amplification. The primers used were sgRNA-specific forward primers and universal reverse primers, their sequences of which are shown in Table 2. The PCR reagent used was PowerPol 2X PCR Mix (purchased from Abclonal, catalog number RK20718). The reaction system was prepared according to the instructions, adding the sgRNA-specific forward primers, universal reverse primers, and the sgRNA backbone template. The PCR reaction program was: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 68℃ annealing and extension for 30 s, 35 cycles; 72℃ final extension for 5 min. After amplification, the PCR products were subjected to agarose gel electrophoresis at a gel concentration of 2%. The band size was confirmed to be approximately 132 bp with no obvious impurities. The target band was then excised and recovered. Grass carp were transcribed according to the instructions of the T7 High Yield RNATranscription Kit (purchased from Vazyme, catalog number TR101). ​In 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.

[0039] Table 2 Grass carp ​ Gene sgRNA in vitro transcription template PCR amplification primers

[0040]

[0041] 1.3 Grass carp ​ Validation of gene sgRNA cleavage efficiency

[0042] Grass carp containing the above-mentioned sgRNA target were amplified by PCR. ​ 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%). ​ As shown in B, grass carp ​ The cleavage efficiency of gene 2 sgRNA is the highest.

[0043] Table 3 Grass carp ​ Primers for amplifying gene sgRNA target fragments

[0044]

[0045] Example 2: ​ Gene knockout grass carp creation

[0046] 2.1 Grass carp embryo microinjection

[0047] Grass carp fertilized eggs were obtained through artificial spawning induction and artificial insemination, and the Cas9 protein was then combined with... ​ The 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. ​ Remove grass carp fry.

[0048] 2.2 Grass carp ​ Mutation identification and screening of high mutation rate F0 generation chimeric individuals

[0049] 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. ​ 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. ​ As shown in Figure A, [the substance] was detected in the injected grass carp fry. ​ 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. ​ Mutation type. For example... ​ As shown in B, a total of 19 species of grass carp were identified. ​ The types of gene mutations were identified, and the proportion of each mutation was statistically analyzed. PAGE electrophoresis was used to analyze the mutations. ​ Further mutation screening was conducted on F0 generation grass carp individuals, and chimeric individuals with a mutation rate of over 50% were selected as... ​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. ​ Individuals with high mutation rates.

[0050] Example 3: ​ Gene knockout grass carp creation

[0051] 3.1 Grass carp embryo microinjection

[0052] Grass carp fertilized eggs were obtained through artificial spawning induction and artificial insemination, and the Cas9 protein was then combined with... ​ 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. ​ Remove grass carp fry.

[0053] 3.2 Grass carp ​ Mutation identification and screening of high mutation rate F0 generation chimeric individuals

[0054] 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. ​ 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... ​ 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. ​ Individuals with high mutation rates accounted for 85% of the total.

[0055] Example 4: F0 generation ​ The survival rate of knocked-out grass carp after viral infection was significantly higher than that of wild-type grass carp.

[0056] The obtained F0 generation ​ 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. ​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. ​ Grass 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... ​ Survival rate of grass carp with high mutation rates. For example... ​ As shown, at the end of the challenge experiment, ​ The survival rate of grass carp with a high mutation rate is about 40% higher than that of wild-type grass carp, indicating that... ​ Knockout grass carp showed a significantly enhanced ability to resist GCRV-II virus infection.

[0057] Example 5: F0 generation ​ The liver lesions in grass carp were relatively mild during the viral challenge process.

[0058] Wild-type grass carp with a body length of 8-10cm and F0 generation were respectively tested. ​ 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... ​ 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. ​ As shown, ​ 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... ​ Knockout grass carp showed a significantly enhanced ability to resist GCRV-II virus infection.

[0059] Example 6: F0 generation ​ Knockout grass carp showed a significantly enhanced innate immune response during the challenge process.

[0060] Wild-type grass carp with a body length of 8-10cm and F0 generation were respectively tested. ​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... ​ Grass carp with a high mutation rate were selected. Liver, spleen, intestine, and kidney tissues were dissected and collected in 1.5 ml centrifuge tubes. RNA was extracted using the Trizol method and stored at -80°C. Reverse transcription was performed according to the instructions of a Vazyme kit (Catalog No. R312) to obtain the first strand of cDNA, which was then stored at -20°C. Real-time quantitative PCR was performed using the SYBR Green qPCR Mix (Catalog No. Q712) to detect the grass carp's innate immune genes. ​ and ​ The level of expression. For example... ​ and ​ As shown, ​ Knock out the liver, spleen, intestines, and kidneys of the grass carp. ​ and ​ The expression levels of [specific ingredient] were significantly higher in grass carp from day 3 to day 7 after challenge, indicating that [the following is likely a continuation of the previous sentence]. ​ Knockout during GCRV-II infection can activate the innate immune antiviral response in grass carp to a greater extent, thereby exerting an anti-hemorrhagic disease effect.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

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; the dhcr7 sequence number of the gene is XM_051900158.

1.

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 targeting sequence of sgRNA is shown as SEQ ID NO. 2, which is 5'-GGAAAGGAGCCGGCACAGTG-3'.

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-inseminate until the grass carp eggs are stored on ice for more than 90 minutes; use a micrometer to measure the size of the 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 targeting sequence thereof is shown as SEQ ID NO. 2, which is 5'-GGAAAGGAGCCGGCACAGTG-3', and the source of the targeting sequence is grass carp.

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

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