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

Jing Han, Yuan Min, Lan Hu, Jie-jie Chen, Shu-Xia Zhang, Xiao-Fan Li, Zhou-Hua Cheng (), Dong-Feng Liu () and Han-Qing Yu ()
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Jing Han: University of Science and Technology of China
Yuan Min: University of Science and Technology of China
Lan Hu: University of Science and Technology of China
Jie-jie Chen: University of Science and Technology of China
Shu-Xia Zhang: Fujian Medical University Union Hospital
Xiao-Fan Li: Fujian Medical University Union Hospital
Zhou-Hua Cheng: University of Science and Technology of China
Dong-Feng Liu: University of Science and Technology of China
Han-Qing Yu: University of Science and Technology of China

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-64010-z

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