Bioscience Methods 2025, Vol.16, No.3, 162-172 http://bioscipublisher.com/index.php/bm 169 a strong ability to invade the ecology. If edited snakehead fish with stronger disease resistance or even faster growth are cultivated, if they are not managed well and enter the wild environment, they may have a more competitive advantage than ordinary snakehead fish, thus causing a greater impact on the local ecology (Robinson et al., 2024). In addition, the impact on other species needs to be considered. Edited snakehead fish may affect the predator-prey relationship through the food chain, or hybridize with closely related species to produce environmental consequences. Considerations from the perspective of ecological diversity: Large-scale promotion of disease-resistant edited fish varieties may lead to reduced genetic diversity in farmed populations. Because these varieties have artificially selected dominant alleles, farmers may tend to use a single strain, thereby reducing the diversity of snakehead culture germplasm (Roy et al., 2022). Low diversity poses risks when encountering environmental changes or new diseases. 6.2 Food safety and market acceptance Any gene-edited animal used for food production must ensure that its edible parts are safe for human consumption and recognized by consumers. In terms of food safety, the scientific community generally believes that if gene-edited animals do not introduce exogenous DNA, their products are essentially no different from traditional breeding products (Roy et al., 2022). For edited snakehead, a case-by-case assessment is required. If the change is only to knock out a snakehead gene, the resulting fish meat does not contain new substances and is theoretically as safe as ordinary snakehead. If the editing introduces exogenous genes (such as antimicrobial peptides), the presence of exogenous proteins in the fish meat and the risk of allergies need to be assessed. Alligator antimicrobial peptides are small molecule polypeptides that may be inactivated during cooking and degraded in the digestive system. They generally do not cause toxicity or allergies (Puthumana et al., 2024). In terms of market and public acceptance, this is a more complicated issue than scientific safety. Many consumers still have doubts or resistance to "genetically modified foods." Although gene editing is different from GMOs, many countries have adjusted their regulations to exclude gene-edited products that do not contain exogenous DNA from GMO regulation, but it will take time for the public to understand it. 6.3 Potential impact of off-target effects and imbalanced repair mechanisms The safety of gene editing technology itself is also an issue that must be taken seriously, of which off-target effects and imbalanced repair mechanisms are two major concerns. Off-target effects refer to the possibility that CRISPR/Cas9 may also produce mutations at other locations in the genome outside the target site. If these mutations fall on key genes, they may cause unexpected traits or health problems (Zhu et al., 2024). In addition to off-target effects, another concern is the imbalance of DNA repair mechanisms. After Cas9 produces double-strand breaks, cells repair the breaks through non-homologous end joining (NHEJ) or homologous recombination (HDR). NHEJ is prone to indel mutations, while HDR can be accurately repaired when there is a template. Large-scale gene editing may trigger stress of DNA damage response in cells, especially in the early stages of embryonic development, when a large number of cells undergo repair at the same time, which may lead to abnormal embryonic development or cell aging. 7 Application Prospects and Future Research Directions 7.1 Establish a database of disease resistance traits and a functional gene resource library for snakehead fish In order to better utilize gene editing to improve the disease resistance of snakehead fish, it is necessary to consolidate basic research and establish a complete disease resistance phenotype and genotype database. Specifically, on the one hand, it is necessary to collect disease resistance phenotypic data of different snakehead fish strains and individuals through large-scale breeding experiments, including survival rate, morbidity, and degree of lesions under different pathogens. In conjunction with genotyping, disease resistance-related genetic markers can be located. This is actually similar to the family challenge test in traditional breeding, but it should be upgraded to a database containing genomic information (Fraslin et al., 2020). On the other hand, existing and future snakehead fish genome and transcriptome data should be integrated to establish a special "snakehead fish disease resistance gene database". It includes the identified snakehead fish immune-related gene sequences, expression profiles, functional annotations, literature evidence, etc. There are fish immune gene databases abroad
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