Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9

• Main Authors: Hirano, Seiichi (Author), Abudayyeh, Omar O. (Author), Gootenberg, Jonathan S (Author), Horii, Takuro (Author), Ishitani, Ryuichiro (Author), Hatada, Izuho (Author), Zhang, Feng (Author), Nishimasu, Hiroshi (Author), Nureki, Osamu (Author)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences (Contributor), McGovern Institute for Brain Research at MIT (Contributor)
• Format: Article
• Language: English
• Published: Springer Science and Business Media LLC, 2019-11-08T21:59:25Z.
• Subjects: Article
• Online Access: Get fulltext

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets bearing a protospacer adjacent motif (PAM) and complementarity to an RNA guide. Unlike other Cas9 orthologs, Corynebacterium diphtheriae Cas9 (CdCas9) recognizes the promiscuous NNRHHHY PAM. However, the CdCas9-mediated PAM recognition mechanism remains unknown. Here, we report the crystal structure of CdCas9 in complex with the guide RNA and its target DNA at 2.9 Å resolution. The structure reveals that CdCas9 recognizes the NNRHHHY PAM via a combination of van der Waals interactions and base-specific hydrogen bonds. Moreover, we find that CdCas9 exhibits robust DNA cleavage activity with the optimal 22-nucleotide length guide RNAs. Our findings highlight the mechanistic diversity of the PAM recognition by Cas9 orthologs, and provide a basis for the further engineering of the CRISPR-Cas9 genome-editor nucleases. Keyword: DNA restriction-modification enzymes; X-ray crystallography
National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)
National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)