Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 31036811.
- Also identified by DOI 10.1038/s41467-019-09741-6 and PMC identifier 6488586.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
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.
Medical subject headings
- CRISPR-Associated Protein 9
- DNA Restriction-Modification Enzymes