Bioscience Methods 2025, Vol.16, No.3, 162-172 http://bioscipublisher.com/index.php/bm 167 than 20 species of fish, covering multiple target traits such as growth, muscle, pigment, reproduction and disease resistance (Ferdous et al., 2022). Specifically for disease resistance, CRISPR technology has shown unique advantages: it can directly knock out negative regulatory genes that restrict disease resistance, or accurately repair/introduce favorable mutations, thereby significantly improving the disease resistance of fish (Wang and Cheng, 2023). For species such as snakehead, the introduction of CRISPR technology will open a new path for rapid improvement of disease resistance. 4.2 Key technical paths for gene editing in snakehead To apply CRISPR/Cas9 technology to snakehead and achieve effective gene editing, it is necessary to overcome the challenges of its reproductive biology and early embryo manipulation and establish a complete set of technical paths. (1) Obtain fertilized eggs and fertilize in vitro. Snakehead gametes can be obtained through artificial induction of spawning, and fertilized eggs can be obtained by in vitro fertilization. Due to the small diameter of snakehead eggs and the tough egg membrane, attention should be paid to the fertilization time window and the maintenance of egg activity. (2) Microinjection of editing elements: Cas9 protein or mRNA encoding it is mixed with the designed gRNA to form an editing reagent, which is injected into the embryonic cells of snakehead fertilized eggs under a microscope using a microneedle (Ou et al., 2023). (3) Embryo hatching and screening: The injected fertilized eggs are hatched at a suitable temperature and develop into F0 generation fry. For those with a high mutation rate, they can be cultured to maturity (Figure 3) (Roy et al., 2022). (4) Establishing homozygous mutant lines: Individuals carrying germline mutations in the F0 generation are mated as broodstock, and some homozygous mutant offspring individuals can be obtained in the F1 generation. For snakehead carp, similarly, 1 to 2 generations of breeding are required to obtain stable knockout or knock-in lines (Zhao et al., 2021). (5) Phenotypic verification: After obtaining homozygous gene-edited snakehead carp, disease resistance can be evaluated. (6) Expansion and evaluation. If the gene editing effect is ideal, the breeding population can be expanded under isolation conditions to evaluate its production traits such as growth and reproduction, as well as environmental adaptability. Snakehead carp is a fast-growing fish that can reach the evaluation body length within a few months. Figure 3 A simple roadmap of general methodology for CRIPSR/Cas genome editing in aquaculture and fisheries (Adopted from Roy et al., 2022) Image Caption: The target gene has to be selected after searching the genome database of candidate species. The sgRNA has to be designed with the help of sgRNA-designing tools, and then, the sgRNA oligo has to be synthesized. For target-specific cleavage, the sgRNA and cas9 mixture needs to be delivered to the newly fertilized embryo at a one-cell stage by microinjection or similar methods. The final step is the assessing the genome-editing results and application stage that includes mutagenesis analysis, the selection of mutants, crossing with wild population and production of a specific mutant line, the evaluation of CRISPR-induced mutation associated phenotyp(s), and the establishment of new varieties with improved values in aquaculture (Adopted from Roy et al., 2022)
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