Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease

Article

关键词: RNA;    CRISPR;    SPECIFICITY;    SYSTEM;    REFINEMENT;    DROSOPHILA;   

DOI  :  10.1038/nature13579

来源: SCIE

【 摘 要 】

The CRISPR-associated protein Cas9 is anRNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guideRNA(1,2). Cas9 hase merged as a versatile molecular tool for genome editing and gene expression control(3). RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA(1,4-6). Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxyterminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

【 授权许可】

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