Format

Send to

Choose Destination
Science. 2016 Feb 19;351(6275):867-71. doi: 10.1126/science.aad8282. Epub 2016 Jan 14.

Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
2
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
3
Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
4
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
5
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA. Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. doudna@berkeley.edu enogales@lbl.gov.
6
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA. Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA. Department of Chemistry, University of California, Berkeley, CA 94720, USA. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. doudna@berkeley.edu enogales@lbl.gov.

Abstract

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.

PMID:
26841432
PMCID:
PMC5111852
DOI:
10.1126/science.aad8282
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center