Enhancing genome editing efficiency in goldfish (Carassius auratus) through utilization of CRISPR-Cas12a (Cpf1) temperature dependency.

The CRISPR/Cas technology has demonstrated revolutionary potential across various fields, including agriculture, medicine, and food safety detection. However, the utility of CRISPR/Cas12a, a particularly promising gene-editing tool, is constrained by its temperature sensitivity, limiting its application in low-temperature environments. In this study, we developed a gene-editing technique based on the CRISPR/Cas12a system in the poikilothermic species goldfish Carassius auratus. We systematically evaluated the editing efficiencies of LbCas12a and AsCas12a on the tyrosinase (tyr) gene under varying temperature conditions. Our results revealed a pronounced temperature dependence of Cas12a, with elevated temperatures markedly enhancing its editing activity, particularly for AsCas12a. A brief one-hour high-temperature treatment was sufficient to achieve effective gene disruption, underscoring CRISPR/Cas12a as a rapid and efficient gene-editing tool. Temperature was utilized as a conditional trigger for Cas12a-mediated gene knockout, enabling precise modulation of gene disruption at specific embryonic developmental stages. Whole-genome resequencing of the mutants confirmed the absence of off-target effects, further emphasizing the precision of this editing process. These findings indicated that CRISPR/Cas12a represented a viable alternative to the widely utilized CRISPR/Cas9 system and could be applied in conjunction, thereby expanding the potential applications of gene-editing technologies.