Bioscience Methods 2025, Vol.16, No.3, 162-172 http://bioscipublisher.com/index.php/bm 168 4.3 Potential targets for gene editing for disease resistance Editing targets for improving disease resistance can be divided into two categories: one is to enhance genes related to immune defense, and the other is to weaken genes related to susceptibility pathways. For the former, the disease resistance of fish can be improved by gene editing knock-in or activation (Gutási et al., 2023). For example, it is possible to consider introducing exogenous antimicrobial peptide genes into the snakehead genome so that it can express broad-spectrum antimicrobial substances at high levels when infected. This is similar to the idea of introducing antimicrobial peptide genes into crocodiles in catfish (Ferdous et al., 2022). For the second type of target, it is to knock out some negative regulatory factors or pathogen receptors to reduce pathogen infection and pathogenic processes. For example, the receptor genes required for viruses to enter host cells in snakehead fish can be knocked out first. If a virus receptor on the cell membrane of snakehead fish is found, it will be difficult for the virus to invade after knocking it out, thereby greatly improving the antiviral ability. Similar cases have been achieved in grass carp (Zhu et al., 2024). In addition, it is possible to consider knocking out genes in the immune suppression pathway. The normal functions of certain genes can inhibit immune responses or promote pathogen escape, and knocking them out can remove the "brakes" on immunity. 5 Case Study and Prospect of Gene-Edited Snakehead Breeding for Disease Resistance 5.1 Analysis of successful cases of gene editing for disease resistance in related fish Although there are no publicly reported cases of disease-resistant gene editing breeding for snakehead, some successful experiences of other aquatic fish can be used for reference. In farmed salmon, infectious pancreatic necrosis (IPN) was once a devastating viral disease, but it has been successfully controlled using traditional breeding and modern genomic methods. The key is the discovery of a major QTL on chromosome 26 of salmon that determines the fish's resistance to IPNV (Pavelin et al., 2021). Inspired by this, the team is trying to introduce the natural disease-resistant mutant Nae1 allele into susceptible salmon strains through CRISPR. American researchers creatively adopted the "enhanced defense" strategy to improve catfish's resistance to bacterial pathogens. Professor Dunham's team used CRISPR/Cas9 to insert the alligator-derived antimicrobial peptide gene Cath into catfish embryos and knocked it into the catfish growth hormone receptor gene site (Ferdous et al., 2022; Ye et al., 2025). This study proves that the idea of cross-species introduction of disease-resistant genes combined with gene editing is feasible, which not only improves the disease resistance of the fish itself, but also ensures biosafety from an ecological perspective. 5.2 Prediction of potential successful cases of disease-resistant gene editing in snakehead fish Nocardia is one of the top killers of snakehead fish farming. It is conceivable to improve the resistance of snakehead fish to this disease through gene editing. One feasible solution is to edit both the infection strategy of Nocardia and the immune response of snakehead fish. There is currently no prevention and treatment for the rhabdovirus SHVV, and gene editing can provide an innovative approach (Michael et al., 2024). It is possible to consider editing the antiviral natural immune pathway of snakehead fish to quickly eliminate the virus. For example, using CRISPR activation technology (CRISPRa) to continuously activate the expression of type I interferon genes or downstream interferon-stimulated genes (such as Mx and PKR) in snakehead fish, the fish is in a "warning" state, and once the virus invades, its replication can be inhibited. Parasitic problems such as wheelworms in snakehead aquaculture are also expected to be alleviated through gene editing. One strategy is to make the surface of snakehead fish (skin, gills) form characteristics that are not conducive to the attachment of parasites. On the other hand, it can also enhance the immune recognition ability of snakehead fish to parasite antigens. With the improvement of CRISPR multiple editing efficiency reported in current literature (successful editing of 45 sites at a time has been achieved in zebrafish, etc.), this "one-step multi-target" solution is technically feasible (Hallerman et al., 2021; Ferdous et al., 2022). If the produced snakehead fish can show excellent survival rate in both bacterial and viral attack tests, it will be a revolutionary new strain. 6 Risk Assessment and Ethical Considerations of Disease Resistance Gene Editing 6.1 Ecological safety issues While gene editing snakehead fish improves disease resistance, it also raises concerns and discussions about the ecological environment. Snakehead fish itself is a top predator fish, and once it escapes to non-local waters, it has
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