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TitleStructures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.
Journal, issue, pagesScience, Vol. 351, Issue 6275, Page 867-871, Year 2016
Publish dateFeb 19, 2016
AuthorsFuguo Jiang / David W Taylor / Janice S Chen / Jack E Kornfeld / Kaihong Zhou / Aubri J Thompson / Eva Nogales / Jennifer A Doudna /
PubMed AbstractBacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding ...Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.
External linksScience / PubMed:26841432 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution3.4 - 6.0 Å
Structure data

EMDB-3276:
Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
Method: EM (single particle) / Resolution: 4.5 Å

EMDB-3277:
Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
Method: EM (single particle) / Resolution: 6.0 Å

PDB-5f9r:
Crystal structure of catalytically-active Streptococcus pyogenes CRISPR-Cas9 in complex with single-guided RNA and double-stranded DNA primed for target DNA cleavage
Method: X-RAY DIFFRACTION / Resolution: 3.4 Å

Chemicals

ChemComp-SO4:
SULFATE ION

Source
  • Streptococcus pyogenes (bacteria)
  • lambda (unknown)
  • streptococcus pyogenes serotype m1 (bacteria)
  • streptococcus pyogenes mgas8232 (bacteria)
  • lambdapapillomavirus 1
KeywordsHYDROLASE/DNA/RNA / CRISPR / Cas9 / R-loop / genome engineering / HYDROLASE-DNA-RNA complex

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