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标题：Mg ²⁺-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage
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作者：Heyjin Son ; Jaeil Park ; Injoo Hwang 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：49
DOI：10.1073/pnas.2113747118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance CRISPR-Cas12a has emerged as attractive molecular scissors alternative to Cas9 owing to its unique features including fewer off-target effects, an alternative protospacer-adjacent motif sequence, pre-CRISPR RNA processing activity, and indiscriminate single-stranded DNase activity. However, despite these advantages, Cas12a has not been well utilized as recently reported base and prime editors because it does not have complete nickase variants, unlike Cas9. In this study, we provide a thorough understanding of the mechanisms that govern the generation of complete double-stranded DNA breaks by the single catalytic site of Cas12a using single-molecule fluorescence assays to improve our ability to develop a rational design for more potently engineered Cas12a including the nickase form. This would extend the range of genome editing applications of Cas12a. CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg 2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg 2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.
关键词：CRISPR-Cas12a; genome editing; single-molecule FRET; DNA cleavage; metal ion