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

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

SUBMITTER: Zeng Y 

PROVIDER: S-EPMC5758904 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature