A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.
A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.
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DOI：
10.1093/nar/gkad636
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发表时间：
2023-09-08
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
--
作者：
CRISPR相关蛋白如Cas9和Cas 12 a是可编程的RNA引导的核酸酶，已成为基因组操作和分子诊断的强大工具。然而，这些酶倾向于切割在RNA指导序列和DNA原型间隔区之间含有错配的脱靶序列。与Cas9相比，Cas 12 a已经显示出对前间区序列邻近基序（PAM）远端错配的独特敏感性，并且Cas 12 a增强的靶辨别的分子基础是非常感兴趣的。在这项研究中，我们使用定点自旋标记，荧光光谱和酶动力学的组合研究了Cas 12 a靶标识别的机制。在完全匹配的RNA引导下，数据显示了DNA解缠绕状态和DNA配对的类RNA状态之间的内在平衡。使用脱靶RNA引导物和预切口DNA底物的实验将PAM-远端DNA解旋平衡鉴定为DNA切割的第一步之前的错配感测检查点。这一发现揭示了Cas 12 a的独特靶向机制，并可能更好地为基于CRISPR的生物技术发展提供信息。
CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.
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