6joo

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

Structural highlights

6joo is a 4 chain structure with sequence from Corynebacterium diphtheriae and Corynebacterium diphtheriae NCTC 13129. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 2.9Å
Ligands:EDO, MSE, ZN
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

CAS9_CORDI CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA) (PubMed:25830891). Cas9 recognizes the protospacer adjacent motif (PAM) in the CRISPR repeat sequences to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs (PubMed:25830891). PAM recognition is also required for catalytic activity (By similarity).[UniProtKB:Q99ZW2][1]

Publication Abstract from PubMed

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 A 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.

Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9.,Hirano S, Abudayyeh OO, Gootenberg JS, Horii T, Ishitani R, Hatada I, Zhang F, Nishimasu H, Nureki O Nat Commun. 2019 Apr 29;10(1):1968. doi: 10.1038/s41467-019-09741-6. PMID:31036811[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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Citations
6 reviews cite this structure
CRISPR technologies and the search for the PAM-free nuclease.
Collias et al. (2021)
5 more
14 other articles
No citations found

See Also

References

  1. ↑ Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015 Apr 9;520(7546):186-91. PMID:25830891 doi:10.1038/nature14299
  2. ↑ Hirano S, Abudayyeh OO, Gootenberg JS, 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. 2019 Apr 29;10(1):1968. doi: 10.1038/s41467-019-09741-6. PMID:31036811 doi:http://dx.doi.org/10.1038/s41467-019-09741-6

Contents


PDB ID 6joo

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