R-loop formation and conformational activation mechanisms of Cas9

Article

关键词: RNA-GUIDED CAS9;    MULTIPLE SEQUENCE ALIGNMENT;    CRYSTAL-STRUCTURE;    TARGET CLEAVAGE;    DNA CLEAVAGE;    COMPLEX;    ENDONUCLEASE;    RECOGNITION;   

DOI  :  10.1038/s41586-022-05114-0

来源: SCIE

【 摘 要 】

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage(1-)(3). The programmable activity of Cas9 has been widely utilized for genome editing applications(4-6), yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcuspyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodatesthe distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

【 授权许可】

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