A method for establishing an anti-nerve necrosis virus grouper population

Abstract

This invention relates to a method for establishing a grouper anti-neural necrosis virus (NSN) family based on an engineered exosome delivery CRISPR / Cas9 system. First, a CRISPR / Cas9 ribonucleoprotein (RNP) complex is pre-assembled in vitro based on the grouper host gene NLRX1. Simultaneously, grouper reproductive stem cells are collected, and the cytoplasm and cell membrane components are separated. A transmembrane peptide targeting the fertilized egg is then embedded into the cell membrane. Subsequently, the membrane components, cytoplasm, ATP regeneration system, and the RNP complex are mixed for a recombination reaction. After purification, engineered exosomes loaded with CRISPR / Cas9 are obtained. Finally, these exosomes are mixed with grouper fertilized eggs and co-incubated to achieve non-invasive gene editing. After incubation, screening, and cultivation, an NSN-resistant population is obtained, and a stable genetic family is established.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for establishing a grouper population resistant to nerve necrosis virus. Background Technology

[0002] Grouper neuronecrosis virus disease, commonly known as "black body disease," is prevalent in most marine aquaculture areas worldwide, particularly in grouper farming areas in southern China (such as Guangdong and Hainan). This virus can harm grouper larvae, juveniles, and adults, with a mortality rate as high as 100% once infected. Infected fish exhibit blackening of body color, spiral swimming, abdominal swelling, swim bladder congestion, and cloudy, bulging eyes. Dissection reveals vacuolation of brain cells and retinal tissue. The virus damages the fish's nervous system, reduces immunity, and makes them susceptible to secondary bacterial and parasitic infections, leading to difficult treatment and severe losses for fish farmers. The key to controlling grouper neuronecrosis virus lies in "source control + environmental stability + immune enhancement." Currently, there is no cure or specific drug; prevention relies mainly on comprehensive management to reduce the risk of infection.

[0003] Gene editing technologies (such as the CRISPR / Cas9 system) have become important tools for breeding disease-resistant aquatic animals. Their basic principle is to block the life cycle of pathogens by targeting and modifying key genes in the host genome that viral replication depends on. However, significant technical bottlenecks remain in the application of this technology in marine fish such as grouper.

[0004] Grouper eggs, due to their high yolk content and fragile membrane structure, are easily damaged by traditional microinjection methods. Current research indicates that pearl grouper (… Epinephelus lanceolatus ♂× E. fuscoguttatus ♀) After microinjection, the survival rate of fertilized eggs decreases, and the effective operation window is within 30 minutes after fertilization, which is insufficient to meet the needs of large-scale breeding. Although non-invasive delivery methods (such as PEI nanoparticles and liposome encapsulation) have made progress in grouper, existing delivery systems still lack tissue specificity, with a penetration efficiency of less than 20% in grouper fertilized eggs, and cannot effectively overcome the multi-layered egg membrane barrier of the embryo. For example, although the carboxyl fluorescent quantum dot-loaded gene editing system can improve delivery efficiency, the universal sgRNA it relies on is not optimized for the grouper virus replication mechanism and cannot effectively target essential viral genes (such as LGP2 and TBLR1).

[0005] More critically, most current research still uses plasmid DNA as the delivery tool for gene editing. Plasmids need to undergo transcription and translation within the cell to form a functional Cas9 protein. This process is delayed, inefficient, and carries the potential risk of random integration of exogenous DNA into the genome, which is particularly concerning in the breeding of edible fish where safety requirements are stringent. Regarding target selection, early studies focused primarily on growth-related genes, while systematic screening and validation of key host factors for viral replication were insufficient.

[0006] Disease resistance traits acquired through gene editing must be efficiently and stably inherited by offspring in order to form a valuable breeding core population. Current technologies often only focus on the editing efficiency of the first generation of individuals, lacking a systematic assessment of the transmission efficiency of germline cells after editing and a stable family selection process design, resulting in many "edited individuals" failing to be transformed into commercially valuable "breeding families".

[0007] In summary, existing technologies suffer from three main shortcomings: first, high invasiveness leads to a sharp drop in embryo survival rates; second, insufficient delivery targeting limits editing efficiency; and third, the lack of specificity in target selection results in poor genetic stability. These problems are interconnected and collectively restrict the industrial application of grouper anti-neural necrosis virus breeding technology. Summary of the Invention

[0008] To address the shortcomings of the prior art, such as low delivery efficiency, poor safety of editing tools, unstable vector sources, and difficulty in guaranteeing genetic stability, this invention discloses a method for establishing a grouper genotype resistant to neuronecrosis virus (NSV) based on an engineered exosome delivery CRISPR / Cas9 system. This method utilizes grouper germline stem cells as a stable source to construct engineered exosomes with target peptides on their surface and internally loaded with CRISPR / Cas9 ribonucleoprotein (RNP) complexes. This achieves efficient, precise, and non-invasive gene editing of grouper fertilized eggs, ultimately obtaining a genotype with stable inherited disease resistance traits.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for establishing a grouper population resistant to neuronecrosis virus includes: designing specific sgRNA based on the NLRX1 gene of grouper, then treating grouper with the specific sgRNA and culturing them to obtain a grouper population resistant to neuronecrosis virus.

[0010] This invention, based on differential transcriptome analysis of grouper infected with neuronecrosis virus (NNV), identifies the mitochondrial NOD-like receptor NLRX1 as a novel and significantly activating gene during NNV infection, building upon the existing screening of known viral replication-promoting factors LGP2 and TBLR. The selection of NLRX1 as the intervention target is based on the fact that, in a grouper brain cell model, knockdown of LGP2, TBLR, and NLRX1, respectively, resulted in the most significant inhibitory effect on NNV replication, reducing viral copy number to the lowest level, confirming that NLRX1 is a key promoter of viral replication. However, the effectiveness and application potential of using this gene as an antiviral breeding target have not yet been revealed and developed by existing technologies.

[0011] Preferably, the sequence of the specific sgRNA is shown in SEQ ID NO:1, and its sequence is 5-AGACACGGAGTCAAACATGG-3.

[0012] This invention belongs to the field of molecular breeding and gene delivery technology for aquatic animals, and specifically relates to a method for establishing a grouper anti-neural necrosis virus family based on an engineered exosome delivery CRISPR / Cas9 system. First, a specific sgRNA was designed based on the grouper host gene NLRX1 and incubated with purified Cas9 protein in vitro to pre-assemble a CRISPR / Cas9 ribonucleoprotein complex. Simultaneously, grouper reproductive stem cells were collected, and a supernatant (III) containing the cytoplasm of the reproductive stem cells and a solution (II) containing cell membrane components were obtained. A transmembrane peptide that specifically targets grouper zygotes was co-incubated with solution II, allowing the transmembrane peptide to intercalate onto the cell membrane, resulting in a functionalized membrane solution (III). Subsequently, supernatant I, solution III, cytoplasm, ATP regeneration system, incubation buffer, and the RNP complex were mixed thoroughly and recombined under suitable conditions. After centrifugation, washing, and purification, engineered exosomes loaded with CRISPR / Cas9 RNPs were obtained. Finally, these engineered exosomes were co-incubated with grouper zygotes to achieve non-invasive gene editing. After incubation, screening, and cultivation, grouper individuals resistant to neuronecrosis virus were obtained, and stable genetic families were established.

[0013] Preferably, the steps include: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA was co-incubated with Cas9 protein to obtain CRISPR / Cas9 ribonucleoprotein complex. B. Stimulate the secretion of exosomes by grouper reproductive stem cells and collect them, while also collecting the cell membrane and cytoplasm. C. Co-incubate the cell membrane with the membrane-penetrating peptide to obtain a functionalized membrane; D. Mix the CRISPR / Cas9 ribonucleoprotein complex, the exosomes, the functionalized membrane, and the cytoplasm to obtain engineered exosomes; E. The engineered exosomes are co-incubated with grouper fertilized eggs to obtain the grouper population resistant to the nerve necrosis virus.

[0014] Preferably, the steps include: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA was co-incubated with Cas9 protein to obtain CRISPR / Cas9 ribonucleoprotein complex. B: B1. Collect grouper germline stem cells, treat them with exosome secretion agonists, and collect the stem cell supernatant and stem cell precipitate after culturing. B2. Centrifuge the stem cell supernatant, collect the precipitate and resuspend it to obtain an exosome-like vesicle suspension. B3. Resuspend the stem cell precipitate, break it down and then centrifuge it to collect the first supernatant; B4. Centrifuge the first supernatant again, collect the precipitate and resuspend it as a cell membrane component, and collect the second supernatant at the same time. B5. Centrifuge the second supernatant again and collect the supernatant as a component of the cytoplasm. C. Co-incubate the cell membrane components with the membrane-penetrating peptides to obtain a functionalized membrane suspension; D. The CRISPR / Cas9 ribonucleoprotein complex, the exosome-like vesicle suspension, the functionalized membrane suspension, the cytoplasmic matrix components, the ATP regeneration system, and the incubation buffer are mixed to obtain engineered exosomes; E. The engineered exosomes are co-incubated with grouper fertilized eggs, and then transferred to an incubation system for normal incubation to obtain the grouper population resistant to the nerve necrosis virus.

[0015] Preferably, the steps include: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA was co-incubated with the Cas9 protein to obtain the CRISPR / Cas9 ribonucleoprotein complex. The molar ratio of the specific sgRNA to the Cas9 protein was 1:2. B: B1. Collect grouper reproductive stem cells, treat them with the exosome secretion agonist, and continue culturing for 36 hours. Then, collect the supernatant of the stem cells and the stem cell precipitate, respectively. The exosome secretion agonist includes one or more of monensin agonists and ionocarrier agonists. B2. The stem cell supernatant is subjected to 300 × [a certain process] sequentially. g Centrifuge for 10 minutes, 2000 × g Centrifuge for 10 minutes to remove cell debris, then centrifuge again at 100,000 × 10⁻⁶. g Centrifuge at ultracentrifugation for 70 minutes, collect the precipitate and resuspend it in PBS to obtain the exosome-like vesicle suspension; B3. Resuspend the stem cell precipitate in a homogenizing solution, and then perform lysis and fragmentation treatment at 1,500 × 10⁻⁶ ppm. g Centrifuge for 10 minutes to separate the contents and collect the first supernatant. B4. The first supernatant is then subjected to 20000 × [a certain process / method / etc.] g Centrifuge for 30 minutes to separate, collect the precipitate and resuspend it in homogenate to obtain the cell membrane component, and collect the second supernatant at the same time; B5. The second supernatant is then subjected to 60000 × g Centrifuge for 60 minutes to separate, and collect the supernatant as a cytoplasmic matrix component; C. Co-incubate the cell membrane component with the membrane-penetrating peptide to obtain the functionalized membrane suspension; the mass ratio of the cell membrane component to the membrane-penetrating peptide is 50~100:1. D. The CRISPR / Cas9 ribonucleoprotein complex, the exosome-like vesicle suspension, the functionalized membrane suspension, the cytoplasmic matrix components, the ATP regeneration system, and the incubation buffer are mixed, and then processed by electroporation, followed by centrifugation, washing, and purification to obtain engineered exosomes; E. The engineered exosomes are co-incubated with grouper fertilized eggs, and then transferred to an incubation system for normal incubation to obtain the grouper population resistant to the nerve necrosis virus; the ratio of the number of engineered exosomes to grouper fertilized eggs is 1:5 to 20.

[0016] Preferably, the steps include: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA and the Cas9 protein were co-incubated in RNP assembly buffer at room temperature for 15 minutes to obtain the CRISPR / Cas9 ribonucleoprotein complex. The molar ratio of the specific sgRNA to the Cas9 protein was 1:2. Preparation of CRISPR / Cas9 ribonucleoprotein (RNP) complex: Based on the grouper host gene NLRX1, a specific sgRNA was designed and synthesized. The chemically synthesized sgRNA and purified high-fidelity Cas9 protein were incubated in vitro at an appropriate molar ratio to pre-assemble a CRISPR / Cas9 ribonucleoprotein complex with editing activity.

[0017] B: B1. Collect grouper reproductive stem cells, treat them with the exosome secretion agonist, and continue culturing for 36 hours. Collect the stem cell supernatant and the stem cell precipitate separately. The exosome secretion agonist includes an ion carrier type agonist with a concentration of 1 μM to 10 μM and a treatment time of 24 to 48 hours. When using ion-carrier agonists, the concentration is 1 μM to 10 μM, and the treatment time is 24 to 48 hours.

[0018] B2. The stem cell supernatant is subjected to 300 × [a certain process] sequentially. g Centrifuge for 10 minutes, 2000 × g Centrifuge for 10 minutes to remove cell debris, then centrifuge again at 100,000 × 10⁻⁶. g Centrifuge at ultracentrifugation for 70 minutes, collect the precipitate and resuspend it in PBS to obtain the exosome-like vesicle suspension; B3. Resuspend the stem cell precipitate in a homogenizing solution, perform lysis and fragmentation treatment, and then incubate at 4°C and 1500 × 10⁻⁶ °C. g Centrifuge for 10 minutes to separate the cells and collect the first supernatant. The cells can be fully lysed by mechanical disruption (such as nitrogen cavitation or ultrasound) or chemical permeation to obtain cell lysate.

[0019] B4. To obtain a purer cytoplasmic matrix, the first supernatant was further purified at 20,000 × 10⁻⁶. g Centrifuge for 30 minutes to separate, collect the precipitate and resuspend it in homogenate to obtain the cell membrane component, and collect the second supernatant at the same time; B5. The second supernatant is then subjected to 60000 × g Centrifuge for 60 minutes to separate, collect the supernatant as the cytoplasmic matrix component, and store at -80℃ for later use; C. The cell membrane component and the transmembrane peptide are slowly shaken at 4°C and co-incubated for 12-16 hours to allow the transmembrane peptide to stably integrate into the cell membrane lipid bilayer through hydrophobic interactions, thereby obtaining the functionalized membrane suspension; the mass ratio of the cell membrane component to the transmembrane peptide is 50-100:1. The membrane-penetrating peptide is a membrane-penetrating peptide that can specifically target grouper fertilized eggs. It can be selected from commonly used research membrane-penetrating peptides such as Transactivator of transcription (MedChemExpress (MCE); Cat. No.: HY-P0282). The incubation mass ratio of the peptide to the cell membrane component is 1:50 to 1:100, the incubation temperature is 4℃, and the incubation time is 12-16 hours. D. Mix the CRISPR / Cas9 ribonucleoprotein complex, the exosome-like vesicle suspension, the functionalized membrane suspension, the cytoplasmic matrix components, the ATP regeneration system, and the incubation buffer. The reaction solution was incubated in a metal bath (e.g., 30°C) for 15–30 minutes to promote the spontaneous reorganization and repair of functionalized membranes and vesicles under ATP-powered conditions. E. The engineered exosomes and grouper fertilized eggs are co-incubated at 24-30°C for 30-90 minutes to achieve non-invasive gene editing of the fertilized eggs, which are then transferred to an incubation system for normal incubation. The hatched individuals are screened to obtain the grouper population resistant to the nerve necrosis virus; the ratio of the engineered exosomes to the grouper fertilized eggs is 1:5-20.

[0020] After incubation, the fertilized eggs are transferred to normal seawater or aquaculture water for further incubation and rearing. During the juvenile stage, individuals with successfully edited NLRX1 genes are screened and identified using target site PCR amplification and sequencing technology. Positive individuals undergo standardized grouper nerve necrosis virus challenge experiments to assess their disease resistance phenotype. Individuals with strong disease resistance are selected as parents, and the population is expanded using conventional breeding methods. Continuous genotypic and disease resistance phenotype analysis is performed on their offspring to ultimately select a grouper family with stable inheritance of the virus-resistant trait.

[0021] Preferably, in step A, the RNP assembly buffer comprises 20 mM HEPES-pH 7.5 and 150 mM KCl; in step B, the homogenizing solution comprises 250 mM sorbitol, 137 mM sodium chloride, and 10 mM PMSF, and the pH is adjusted to 7.4 with Tris-HCl buffer; in step D, the ATP regeneration system comprises 50 mM ATP, 200 mM GDP-mannose, 400 mM creatine phosphate, 20 mM creatine phosphokinase, 20 mM HEPES-pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, and 5 mM magnesium acetate; the incubation buffer comprises 80 mM potassium chloride, 20 mM calcium chloride, 12.5 mM HEPES-NaOH-pH 7.4, 1.5 mM MgOAc, and 1 mM DTT.

[0022] Preferably, step B1 includes: reviving and culturing the established pearl giant grouper reproductive stem cell line, and passage culturing it in an incubator containing 5% CO2 at 28°C; when the cells grow to 80% confluence, replacing it with serum-free medium containing 15 μM GW4869, and continuing to culture for 36 hours, collecting the stem cell supernatant and the stem cell precipitate respectively.

[0023] Preferably, the grouper includes, but is not limited to, one or more of the following: pearl grouper, giant grouper, spotted grouper, red-spotted grouper, green grouper, red grouper, and oblique-banded grouper.

[0024] A group of grouper resistant to nerve necrosis virus obtained by the above-mentioned method for establishing such a grouper population.

[0025] Compared with the prior art, implementing the present invention has the following beneficial effects: 1. This invention relates to a method for establishing a grouper anti-neural necrosis virus (NSN) family based on an engineered exosome delivery CRISPR / Cas9 system. First, a CRISPR / Cas9 ribonucleoprotein (RNP) complex is pre-assembled in vitro based on the grouper host gene NLRX1. Simultaneously, grouper reproductive stem cells are collected, and the cytoplasm and cell membrane components are separated. A transmembrane peptide targeting the fertilized egg is then embedded into the cell membrane. Subsequently, the membrane components, cytoplasm, ATP regeneration system, and the RNP complex are mixed for a recombination reaction. After purification, engineered exosomes loaded with CRISPR / Cas9 are obtained. Finally, these exosomes are mixed with grouper fertilized eggs and co-incubated to achieve non-invasive gene editing. After incubation, screening, and cultivation, an NSN-resistant population is obtained, and a stable genetic family is established.

[0026] 2. This invention achieves a dual innovation in gene editing tools and delivery vectors, significantly improving editing efficiency and biosafety. Compared to existing technologies that deliver CRISPR / Cas9 plasmid DNA, this invention uses a pre-assembled Cas9 protein / sgRNA ribonucleoprotein (RNP) complex as the editing tool, which is then loaded into engineered exosomes for delivery. Once inside the cell, the RNP rapidly exerts its editing function, avoiding the delays and uncertainties associated with intracellular transcription and translation of plasmids, resulting in a faster and more efficient editing process. Simultaneously, this strategy completely avoids the potential risk of random integration of exogenous plasmid DNA into the host genome, aligning with the safety trend of "DNA-free" editing and providing higher biosafety guarantees for cultivating safe new aquatic varieties. Furthermore, the use of stably cultured grouper reproductive stem cells as the exosome source replaces the reliance on large numbers of fertilized eggs in traditional methods, ensuring the stability and scalability of raw material supply.

[0027] 3. This invention overcomes the bottleneck of low efficiency in non-invasive delivery by endowing the delivery vector with active targeting capabilities. Unlike existing technologies that rely on random endocytosis or passive diffusion of exosomes, this invention embeds a membrane-penetrating peptide on the membrane surface of engineered exosomes that can specifically recognize and bind to the receptor on the fertilized grouper egg membrane. This design transforms the exosome from "passive contact" to "active target seeking," greatly improving the efficiency of the gene editing system's specific uptake and internalization by the fertilized egg through receptor-ligand-mediated memory interaction. This active targeting strategy effectively solves the problem of low delivery efficiency caused by the fertilized grouper egg membrane barrier, laying a key foundation for achieving efficient and precise gene editing.

[0028] 4. This invention provides a systematic solution from efficient editing to stable breeding, with significant prospects for industrial application. This solution is not an isolated technological improvement, but rather a complete technical system integrating "innovative target (NLRX1) - advanced tool (RNP) - intelligent carrier (targeted exosomes) - stable source (reproductive stem cells) - family selection." By using passageable reproductive stem cell lines, standardized and large-scale preparation of engineered exosomes is achieved, overcoming the shortcomings of traditional methods such as large batch variations and difficulty in scale-up. Finally, through genotypic screening and standardized antiviral phenotype verification of edited individuals, and systematic breeding work, it is ensured that disease resistance traits can be stably inherited to offspring, thereby truly establishing a grouper disease-resistant family with breeding value, providing a reliable technical platform for the industrial promotion of aquatic disease-resistant breeding. Attached Figure Description

[0029] Figure 1 In the diagram, A shows a volcano diagram of differentially expressed genes between grouper infected with neuronecrosis virus and healthy grouper; B shows the transfection of siRNAs targeting LGP2, TBLR, and NLRX1 into grouper brain cells to interfere with their endogenous expression. After successful interference, the cells were inoculated with neuronecrosis virus, and the viral copy number was measured 24 hours later.

[0030] Figure 2 This is a flowchart of the method described in this invention.

[0031] Figure 3 In the table, A shows the detection of Cas9 protein in RNP-loaded and engineered exosomes and unloaded engineered exosomes; B is a schematic diagram of fluorescence microscopy observation results of pearl grouper larvae co-incubated with RNP-loaded engineered exosomes as described in Example 1 and incubated without engineered exosomes; C shows the editing efficiency detected using the T7EI method; and D shows the survival rate of pearl grouper after NLRX1 gene fragment mutation in the grouper as described in Example 1 and subsequent infection with neuronecrosis virus. Detailed Implementation

[0032] The present invention is further illustrated by the following examples, but these are not intended to limit the invention. Specific experimental conditions and methods not specified in the following examples are generally conventional methods well known to those skilled in the art.

[0033] Example 1: A method for establishing an antiviral grouper family, comprising the following steps: (1) Transcriptome analysis of differentially expressed genes in grouper infected with neuronecrosis virus and healthy grouper revealed differential expression of NLRX1 ( Figure 1 A).

[0034] (2) Functional verification experiments showed that after interfering with the expression of LGP2, TBLR and NLRX1 in cultured grouper brain cells and infecting them with neuronecrosis virus, NLRX1 knockdown led to the most significant decrease in viral replication level. This result functionally confirms that NLRX1 is a key host factor for neuronecrosis virus replication, and its promoting effect is stronger than that of LGP2 and TBLR. Figure 1 B).

[0035] (3) Preparation of CRISPR / Cas9 RNP complex targeting NLRX1 gene: sgRNA was designed based on the NLRX1 gene of grouper, with the sequence 5-AGACACGGAGTCAAACATGG-3; at the same time, high-fidelity sgRNA was obtained from the expression strain using Ni-NTA affinity chromatography column. Sp Cas9-HF1 protein. The purified Cas9 protein and each sgRNA were incubated at a molar ratio of 1:2 in RNP assembly buffer (20 mM HEPES, 150 mM KCl, pH 7.5) at room temperature for 15 minutes to form the Cas9-sgRNA ribonucleoprotein complex. The complex was then placed on ice for later use. (4) Culture and processing of engineered exosome-derived cells: The established pearl grouper reproductive stem cell line was revived and cultured, and passaged in an incubator containing 5% CO2 at 28°C. When the cells reached 80% confluence, the medium was replaced with serum-free medium containing 15 μM GW4869 (a neutral sphingomyelinase inhibitor), and cultured for another 36 hours to stimulate exosome secretion. The cell supernatant and cell pellet were then collected for subsequent separation. (5) Cell component separation and membrane functionalization modification: a. Separation of exosomes from the cytoplasm: The cell supernatant collected in step (2) was subjected to 300 × 10⁻⁶ cycles at 4°C. g (10 minutes), 2,000 × g Centrifuge (10 minutes) to remove cell debris, then centrifuge at 100,000 × 10⁻⁶.g Centrifuge at ultracentrifugation for 70 minutes. Resuspend the precipitate in 100 μL of pre-cooled PBS to obtain an enriched exosome-like vesicle suspension; b. Separation of cell membrane from cytoplasm: The cell pellet collected in step (2) was resuspended in pre-cooled homogenate (composition: 250 mM sorbitol, 137 mM NaCl, 10 mM PMSF, 10 mM Tris-HCl, pH 7.4) and lysed on ice using a nitrogen cavitation lysate at 500 psi. The lysis buffer was then incubated at 4°C at 1,500 × 10⁻⁶ ppm. g Centrifuge for 10 minutes to remove intact cells and nuclear precipitate, and then centrifuge the supernatant at 20,000 × 10⁻⁶. g Centrifuge for 30 minutes. The precipitate from this centrifugation is resuspended in homogenate as the cell membrane component; the supernatant is further centrifuged at 60,000 × 10⁻⁶. g Centrifuge for 60 minutes. The supernatant from this centrifugation will be used as the cytoplasmic matrix component and stored at -80℃ for later use. c. Cell membrane functionalization: Take 200 μg (based on membrane proteins) of the cell membrane component obtained in step (3b) and incubate with 4 μg of the targeted transmembrane peptide (externally purchased transactivator of transcription (MedChemExpress (MCE); Cat. No.: HY-P0282)) at 4°C with slow shaking overnight to allow the transmembrane peptide to intercalate onto the membrane; (6) Preparation of RNP-loaded targeted engineered exosomes: Take 50 μL of exosome vesicle suspension obtained in step (3a), 30 μL of functionalized membrane suspension obtained in step (3c), 50 μL of cytoplasmic matrix obtained in step (3b), 10 μL of ATP regeneration system (50 mM ATP, 200 mM GDP-mannose, 400 mM phosphocreatine, 20 U / mL phosphocreatinease, 20 mM HEPES pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, 5 mM magnesium acetate), 20 μL of incubation buffer (80 mM KCl, 20 mM CaCl2, 12.5 mM HEPES-NaOH pH 7.4, 1.5 mM MgOAc, 1 mM DTT), and mix the RNP complex prepared in step (1) (containing 5 μg Cas9 protein and an equimolar amount of sgRNA mixture); (7) Incubation of engineered exosomes with fertilized eggs and verification of gene editing: Freshly fertilized pearl grouper fertilized eggs were collected and engineered exosomes prepared in step (4) were added. The exosomes and fertilized eggs were incubated together at 28°C for 30 minutes, with gentle shaking during the incubation. After incubation, the fertilized eggs were washed three times with fresh seawater and transferred to the incubation system for normal incubation. Figure 2 ).

[0036] Western blot analysis showed that engineered exosomes were successfully loaded with RNP ( Figure 3 A), after successfully loading engineered exosomes onto RNPs and co-incubating them with grouper fertilized eggs, fluorescence microscopy of pearl grouper larvae revealed fluorescence, indicating that the editing tool had entered the grouper larvae. Figure 3 B).

[0037] The NLRX1 gene was detected in grouper treated by the method described in Example 1, and the editing efficiency was detected using the T7EI method. The NLRX1 gene editing efficiency was 71.82%. Figure 3 C).

[0038] The grouper larvae treated by the method described in Example 1 showed significantly enhanced resistance to neuronecrosis virus after 30 days of cultivation, compared with the control group that did not undergo gene editing and whose fertilized eggs developed normally. Figure 3 D). In the control group, the number of grouper larvae dying daily due to infection with neuronecrosis virus was 200-400, while in the experimental group, the number of deaths was less than 10 per day.

[0039] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for establishing an anti-NNV grouper population, characterized by, include: A specific sgRNA was designed based on the NLRX1 gene of grouper, and then grouper were treated with the specific sgRNA and cultured to obtain a grouper population resistant to nerve necrosis virus.

2. The method of claim 1, wherein the anti-nervous necrosis virus grouper population is established by, The sequence of the specific sgRNA is shown in the sequence listing SEQ ID NO:

1.

3. The method of claim 1, wherein the anti-nervous necrosis virus grouper population is established by, Includes the following steps: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA was co-incubated with Cas9 protein to obtain CRISPR / Cas9 ribonucleoprotein complex. B. Stimulate the secretion of exosomes by grouper reproductive stem cells and collect them, while also collecting the cell membrane and cytoplasm. C. Co-incubate the cell membrane with the membrane-penetrating peptide to obtain a functionalized membrane; D. Mix the CRISPR / Cas9 ribonucleoprotein complex, the exosomes, the functionalized membrane, and the cytoplasm to obtain engineered exosomes; E. The engineered exosomes are co-incubated with grouper fertilized eggs to obtain the grouper population resistant to the nerve necrosis virus.

4. The method of claim 1, wherein the anti-nervous necrosis virus grouper population is established by, Includes the following steps: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA was co-incubated with Cas9 protein to obtain CRISPR / Cas9 ribonucleoprotein complex. B： B1. Collect grouper germline stem cells, treat them with exosome secretion agonists, and collect the stem cell supernatant and stem cell precipitate after culturing. B2. Centrifuge the stem cell supernatant, collect the precipitate and resuspend it to obtain an exosome-like vesicle suspension. B3. Resuspend the stem cell precipitate, break it down and then centrifuge it to collect the first supernatant; B4. Centrifuge the first supernatant again, collect the precipitate and resuspend it as a cell membrane component, and collect the second supernatant at the same time. B5. Centrifuge the second supernatant again and collect the supernatant as a component of the cytoplasm. C. Co-incubate the cell membrane components with the membrane-penetrating peptides to obtain a functionalized membrane suspension; D. The CRISPR / Cas9 ribonucleoprotein complex, the exosome-like vesicle suspension, the functionalized membrane suspension, the cytoplasmic matrix components, the ATP regeneration system, and the incubation buffer are mixed to obtain engineered exosomes; E. The engineered exosomes are co-incubated with grouper fertilized eggs, and then transferred to an incubation system for normal incubation to obtain the grouper population resistant to the nerve necrosis virus.

5. The method of claim 4, wherein the anti-nervous necrosis virus grouper population is established by, Includes the following steps: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA was co-incubated with the Cas9 protein to obtain the CRISPR / Cas9 ribonucleoprotein complex. The molar ratio of the specific sgRNA to the Cas9 protein was 1:

2. B： B1. Collect grouper reproductive stem cells, treat them with the exosome secretion agonist, and continue culturing for 36 hours. Then, collect the supernatant of the stem cells and the stem cell precipitate, respectively. The exosome secretion agonist includes one or more of monensin agonists and ionocarrier agonists. B2, the stem cell supernatant is sequentially subjected to 300 x g centrifugation for 10 minutes at 2000 x g centrifugation for 10 minutes to remove cell debris, and then 100000 x g ultracentrifugation for 70 minutes, the precipitate is collected, resuspended with PBS, and the exosome-like vesicle suspension is obtained; B3. Resuspend the stem cell pellet with homogenization fluid, and after disruption and lysis, 1,500 x g Separate by centrifugation for 10 minutes, and collect the first supernatant. B4. centrifuging the first supernatant at 20000 x g for 30 minutes g centrifuging for 30 minutes, collecting the precipitate, resuspending it with the homogenization liquid, as the cell membrane fraction, and collecting the second supernatant; B5. The second supernatant is again centrifuged at 60000 x g for 60 minutes g The supernatant is collected as the cytoplasmic matrix fraction. C. Co-incubate the cell membrane components with the membrane-penetrating peptide to obtain the functionalized membrane suspension; The mass ratio of the cell membrane component to the transmembrane peptide is 50-100:

1. D. The CRISPR / Cas9 ribonucleoprotein complex, the exosome-like vesicle suspension, the functionalized membrane suspension, the cytoplasmic matrix components, the ATP regeneration system, and the incubation buffer are mixed, and then processed by electroporation, followed by centrifugation, washing, and purification to obtain engineered exosomes; E. The engineered exosomes are co-incubated with grouper fertilized eggs, and then transferred to an incubation system for normal incubation to obtain the grouper population resistant to the nerve necrosis virus; the ratio of the number of engineered exosomes to grouper fertilized eggs is 1:5 to 20.

6. The method of claim 4, wherein the anti-nervous necrosis virus grouper population is established by, Includes the following steps: A. Based on the NLRX1 gene of grouper, the specific sgRNA was designed and obtained. The specific sgRNA and the Cas9 protein were co-incubated in RNP assembly buffer at room temperature for 15 minutes to obtain the CRISPR / Cas9 ribonucleoprotein complex. The molar ratio of the specific sgRNA to the Cas9 protein was 1:

2. B： B1. Collect grouper reproductive stem cells, treat them with the exosome secretion agonist, and continue culturing for 36 hours. Collect the stem cell supernatant and the stem cell precipitate separately. The exosome secretion agonist includes an ion carrier type agonist with a concentration of 1 μM to 10 μM and a treatment time of 24 to 48 hours. B2. The stem cell supernatant is subjected to 300 × [a certain process] sequentially. g Centrifuge for 10 minutes, 2,000 × g Centrifuge for 10 minutes to remove cell debris, then centrifuge again at 100,000 × 10⁻⁶. g Centrifuge at ultracentrifugation for 70 minutes, collect the precipitate and resuspend it in PBS to obtain the exosome-like vesicle suspension; B3. The stem cell precipitate was resuspended in a homogenizing solution, subjected to lysis and fragmentation treatment, and then incubated at 4°C and 1,500 × 10⁻⁶ °C. g Centrifuge for 10 minutes to separate the contents and collect the first supernatant. B4. The first supernatant is then subjected to 20,000 × g Centrifuge for 30 minutes to separate, collect the precipitate and resuspend it in homogenate to obtain the cell membrane component, and collect the second supernatant at the same time; B5. The second supernatant is then subjected to a further process at 60,000 × g Centrifuge for 60 minutes to separate, collect the supernatant as the cytoplasmic matrix component, and store at -80℃ for later use; C. The cell membrane component and the membrane-penetrating peptide are slowly shaken at 4°C and co-incubated for 12-16 hours to obtain the functionalized membrane suspension; the mass ratio of the cell membrane component to the membrane-penetrating peptide is 50-100:

1. D. The CRISPR / Cas9 ribonucleoprotein complex, the exosome-like vesicle suspension, the functionalized membrane suspension, the cytoplasmic matrix components, the ATP regeneration system, and the incubation buffer are mixed, and then processed by electroporation, followed by centrifugation, washing, and purification to obtain engineered exosomes; E. The engineered exosomes and grouper fertilized eggs are co-incubated at 24-30°C for 30-90 minutes, and then transferred to an incubation system for normal incubation to obtain the grouper population resisting nerve necrosis virus; the ratio of engineered exosomes to grouper fertilized eggs is 1:5-20.

7. The method for establishing a grouper population against neuronecrosis virus according to claim 6, characterized in that, In step A, the RNP assembly buffer comprises 20 mM HEPES-pH 7.5 and 150 mM KCl; in step B, the homogenizing solution comprises 250 mM sorbitol, 137 mM sodium chloride, and 10 mM PMSF, and the pH is adjusted to 7.4 with Tris-HCl buffer; in step D, the ATP regeneration system comprises 50 mM ATP, 200 mM GDP-mannose, 400 mM creatine phosphate, 20 mM creatine phosphokinase, 20 mM HEPES-pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, and 5 mM magnesium acetate; the incubation buffer comprises 80 mM potassium chloride, 20 mM calcium chloride, 12.5 mM HEPES-NaOH-pH 7.4, 1.5 mM MgOAc, and 1 mM DTT.

8. The method for establishing a grouper population against neuronecrosis virus according to claim 4, characterized in that, Step B1 includes: reviving and culturing the established pearl giant grouper reproductive stem cell line, and passaged it in an incubator containing 5% CO2 at 28°C; when the cells grow to 80% confluence, the medium is replaced with serum-free medium containing 15 μM GW4869, and cultured for another 36 hours before collecting the stem cell supernatant and the stem cell precipitate.

9. The method for establishing a grouper population against neuronecrosis virus according to claim 1, characterized in that, The grouper includes one or more of the following: pearl grouper, giant grouper, spotted grouper, red-spotted grouper, green grouper, red grouper, and oblique-banded grouper.

10. A group of grouper resistant to nerve necrosis virus obtained by the method for establishing a group of grouper resistant to nerve necrosis virus according to claim 1.

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