Optimizing Genome Editing in Mollusks ( <i>Crassostrea gigas</i> ) <i>in Vitro</i> Validation of sgRNA and Identifying Key Factors Influencing Efficiency

CRISPR-Cas9 genome editing holds tremendous potential for accelerating genetic improvements in aquaculture. The success of the CRISPR-Cas9 system relies on the specificity and efficiency of engineered single-guide RNAs (sgRNAs). In this study, we optimized an <i>in vitro</i> validation protocol for sgRNAs to streamline the gene editing process, capitalizing on the limited breeding season of the Pacific oyster (<i>Crassostrea gigas</i>). We evaluated the efficiency of 11 sgRNAs targeting four genes both <i>in vitro</i> and <i>in vivo</i> in <i>C. gigas</i>. In addition, we found that Cas9 protein differs from Cas9 mRNA in gene editing efficiency at various stages of early development. Cas9 protein proved particular efficacy in achieving early and efficient gene knockout, functioning effectively during the first cell division and facilitating biallelic gene knockouts. Statistical analysis showed that in the protein group, the biallelic editing frequency ranged from 12.5% to 57.8%, and the overall editing frequency reached as high as 75-90.6%. The mRNA group exhibited a biallelic editing frequency of 3.1-14.0% and the overall editing frequency spanning 65.6-78.1%. Contrary to expectations, low-temperature incubation (20°C) of oyster embryos prolonged the time window for the first cell division but did not improve gene editing efficiency, likely due to the high temperature sensitivity of Cas9 enzyme activity. Together, this study provides a comprehensive analysis of factors affecting the efficiency of CRISPR-Cas9 gene editing in <i>C. gigas</i>, providing a robust framework for future gene editing endeavors in mollusks and other marine invertebrates.