期刊
NATURE COMMUNICATIONS
卷 7, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/ncomms12778
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资金
- National Science Foundation [PHY-1430124, 1244557]
- National Institutes of Health [GM065367, GM112659]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1244557] Funding Source: National Science Foundation
Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from o0.006 s(-1) to 42 s(-1) upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate o0.006 s(-1)), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.
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