Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
29 04 2019
29 04 2019
Historique:
received:
07
10
2018
accepted:
29
03
2019
entrez:
1
5
2019
pubmed:
1
5
2019
medline:
14
5
2019
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 31036811
doi: 10.1038/s41467-019-09741-6
pii: 10.1038/s41467-019-09741-6
pmc: PMC6488586
doi:
Substances chimiques
DNA Restriction-Modification Enzymes
0
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1968Subventions
Organisme : NIMH NIH HHS
ID : R01 MH110049
Pays : United States
Organisme : NHGRI NIH HHS
ID : RM1 HG006193
Pays : United States
Organisme : NHLBI NIH HHS
ID : DP1 HL141201
Pays : United States
Organisme : NIMH NIH HHS
ID : DP1 MH100706
Pays : United States
Organisme : NHGRI NIH HHS
ID : R01 HG009761
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK097768
Pays : United States
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