6JOO

Crystal structure of Corynebacterium diphtheriae Cas9 in complex with sgRNA and target DNA

Summary for 6JOO

• Entry DOI: 10.2210/pdb6joo/pdb
• Descriptor: CRISPR-associated protein,CRISPR-associated endonuclease Cas9, Guide RNA, Non-Target DNA, ... (7 entities in total)
• Functional Keywords: crispr-cas, cas9, pam, dna endonuclease, ribonucleoprotein, hydrolase-dna-rna complex, hydrolase/dna/rna
• Biological source: Corynebacterium diphtheriae; More
• Total number of polymer chains: 4
• Total formula weight: 151414.24

Authors

Hirano, S.,Ishitani, R.,Nishimasu, H.,Nureki, O. (deposition date: 2019-03-22, release date: 2019-04-17, Last modification date: 2024-11-20)

Primary citation

Hirano, S.,Abudayyeh, O.O.,Gootenberg, J.S.,Horii, T.,Ishitani, R.,Hatada, I.,Zhang, F.,Nishimasu, H.,Nureki, O.
Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.
Nat Commun, 10:1968-1968, 2019

PubMed Abstract: 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.

PubMed: 31036811
DOI: 10.1038/s41467-019-09741-6

Experimental method

X-RAY DIFFRACTION (2.9 Å)