Kim Yuyoung, Choi You Hee, Kim Minji, Jang Yunsu, Lee Sanghwa
Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul 06591; Department of Medical Sciences, Graduate School of The Catholic University of Korea, Seoul 06591, Korea.
Korea Institute of Medical Microrobotics, Gwangju 61011, Korea.
BMB Rep. 2025 Aug;58(8):364-368. doi: 10.5483/BMBRep.2025-0073.
CRISPR-Cas12a proteins are RNA-guided endonucleases classified as type V-A effectors that function similarly to Cas9, but possess distinct biochemical features. Previous studies have reported that compared to Cas9, Cas12a exhibits reduced off-target activity, yet the mechanistic origin of this high specificity remains unclear. In this study, we used singlemolecule fluorescence assays to investigate the kinetic basis for the reduced off-target effects of Cas12a. Introducing double mismatches at various positions within the target DNA enabled systematic analysis of the off-target effects on individual reaction steps in the Cas12a-mediated DNA cleavage reaction: seeding, stable R-loop formation, and DNA cleavage. Our results show that mismatches within a 17 bp PAM-proximal seed region significantly impair stable R-loop formation and subsequent cleavage, whereas mismatches in the PAM-distal region exert minimal or negligible effects. These results suggest that the low off-target tolerance of Cas12a and the resulting high on-target selectivity arise from the high sensitivity of the R-loop formation rate to DNA mismatches in the PAM-proximal region, which strongly correlates with cleavage efficiency. This work establishes R-loop formation as a conformational checkpoint for specific target cleavage, and provides a mechanistic framework to improve the fidelity of genome editing. [BMB Reports 2025; 58(8): 364-368].
CRISPR-Cas12a蛋白是一类RNA引导的核酸内切酶，属于V-A型效应蛋白，其功能与Cas9相似，但具有独特的生化特性。此前的研究报道，与Cas9相比，Cas12a的脱靶活性较低，但其高特异性的机制起源尚不清楚。在本研究中，我们使用单分子荧光测定法来研究Cas12a脱靶效应降低的动力学基础。在靶DNA的不同位置引入双错配，能够系统地分析脱靶效应对Cas12a介导的DNA切割反应中各个反应步骤的影响：起始、稳定R环形成和DNA切割。我们的结果表明，在17 bp的PAM近端种子区域内的错配会显著损害稳定R环的形成和随后的切割，而PAM远端区域的错配影响极小或可忽略不计。这些结果表明，Cas12a的低脱靶耐受性和由此产生的高靶向选择性源于R环形成速率对PAM近端区域DNA错配的高敏感性，这与切割效率密切相关。这项工作将R环形成确立为特定靶标切割的构象检查点，并提供了一个机制框架来提高基因组编辑的保真度。[《BMB报告》2025年；58(8):364 - 368]
