Li Huijuan, Liu Lili, Wang Xiaowen, Zhang Rong, Zhu Hua
Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China.
Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China.
Int J Biol Macromol. 2025 May;305(Pt 2):141142. doi: 10.1016/j.ijbiomac.2025.141142. Epub 2025 Feb 17.
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.
CRISPR/Cas技术已在包括农业、医学和食品安全检测在内的各个领域展现出革命性潜力。然而，CRISPR/Cas12a这一特别有前景的基因编辑工具，因其温度敏感性而受到限制，制约了其在低温环境中的应用。在本研究中，我们在变温动物金鱼（Carassius auratus）中开发了一种基于CRISPR/Cas12a系统的基因编辑技术。我们系统评估了LbCas12a和AsCas12a在不同温度条件下对酪氨酸酶（tyr）基因的编辑效率。我们的结果揭示了Cas12a对温度有显著依赖性，温度升高会显著增强其编辑活性，尤其是对于AsCas12a。短暂的一小时高温处理足以实现有效的基因破坏，这突出了CRISPR/Cas12a作为一种快速高效的基因编辑工具的特性。温度被用作Cas12a介导的基因敲除的条件触发因素，能够在特定胚胎发育阶段精确调控基因破坏。对突变体的全基因组重测序证实不存在脱靶效应，进一步强调了这种编辑过程的精确性。这些发现表明，CRISPR/Cas12a是广泛使用的CRISPR/Cas9系统的一个可行替代方案，并且可以联合应用，从而扩展基因编辑技术的潜在应用范围。
