The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex. Issue 1 (14th November 2017)

• Record Type: Journal Article
• Authors: Zeng, Yan; Cui, Yang; Zhang, Yong; Zhang, Yanruo; Liang, Meng; Chen, Hui; Lan, Jie; Song, Guangtao; Lou, Jizhong
• Is Part Of: Nucleic acids research. Volume 46:Issue 1(2018)
• Page Start: 350
• Page End: 361
• Publication Date: 2017-11-14
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkx1117
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique. Our results show that Cas9–sgRNA complex divides the target DNA into several distinct domains: protospacer adjacent motif, linker, Seed, Middle and Tail. After seed pairing, the Cas9 transiently retains a semi-active conformation and induces the cleavage of either target or non-target strand. smFRET studies demonstrate that an intermediate state exists in prior to the formation of the fully stable R-loop complex. Kinetics analysis of this new intermediate state indicates that the lifetime of this state increases when the base-pairing length of guide-DNA hybrid duplex increases and reaches the maximum at the size of 18 bp. These data provide new insights into the process of R-loop formation and reveal the source of off-targeting in CRISPR/Cas9 system.