The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex

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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.

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Zeng, Y., Cui, Y., Zhang, Y., Zhang, Y., Liang, M., Chen, H., … Lou, J. (2018). The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex. Nucleic Acids Research, 46(1), 350–361. https://doi.org/10.1093/nar/gkx1117

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