DNA 解旋平衡可作为 CRISPR-Cas12a 靶标区分的检查点。
A DNA unwinding equilibrium serves as a checkpoint for CRISPR-Cas12a target discrimination.
机构信息
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
出版信息
Nucleic Acids Res. 2023 Sep 8;51(16):8730-8743. doi: 10.1093/nar/gkad636.
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.
CRISPR 相关蛋白，如 Cas9 和 Cas12a，是可编程的 RNA 指导的核酸酶，已成为基因组操作和分子诊断的强大工具。然而，这些酶容易切割出靶序列中与 RNA 指导和 DNA 原间隔序列之间存在错配的非靶序列。与 Cas9 相比，Cas12a 对原间隔序列邻近基序（PAM）远端错配表现出明显的敏感性，Cas12a 增强的靶标区分的分子基础引起了极大的兴趣。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来研究 Cas12a 靶标识别的机制。在完全匹配的 RNA 向导的情况下，数据显示在 DNA 解链状态和 DNA 配对双链类似状态之间存在固有平衡。使用非靶 RNA 向导和预切口 DNA 底物的实验确定了 PAM 远端 DNA 解链平衡作为 DNA 切割第一步之前的错配感应检查点。这一发现揭示了 Cas12a 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。
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