Tailoring Cas12a functionality with a user-friendly and versatile crRNA variant toolbox

作者：Jing Han, Min Yuan, Lan Hu, Jie‐Jie Chen, Shuxia Zhang, Xiaofan Li, Zhou‐Hua Cheng, Dong‐Feng Liu, Han‐Qing Yu · 发表于：Nature Communications · 年份：2025 · DOI：10.1038/s41467-025-64010-z · 被引用次数：18 · 研究领域：CRISPR and Genetic Engineering、RNA and protein synthesis mechanisms、RNA regulation and disease

Cas12a, with its unique targeting and cleavage activity towards DNA, has been widely applied in gene editing and molecular diagnostics. However, there currently lacks an activity regulation strategy that combines flexibility and simplicity to adapt Cas12a to different demands across various application scenarios. In this study, we present a simple yet effective strategy, wherein we systematically mutate the crRNA direct repeat (DR) sequence to uncover a range of distinct crRNA mutants, which are then compiled into a crRNA toolbox to enable flexible regulation of Cas12a activity. By harnessing the complementarity and synergy between these mutants, we successfully enhance Cas12a performance across various application scenarios. Our crRNA toolbox enables fine-tuned control over expression levels, improves base editing accuracy, enhances transformation and editing efficiency in prokaryote homologous recombination-mediated gene editing, and facilitates rapid, accurate, one-pot, semi-quantitative nucleic acid diagnostics. In summary, the DR sequence mutation strategy provides simple, flexible, and diverse options for Cas12a activity regulation and functional optimization.