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

Fuguo Jiang; David W Taylor; Janice S Chen; Jack E Kornfeld; Kaihong Zhou; Aubri J Thompson; Eva Nogales; Jennifer A Doudna

doi:10.1126/science.aad8282

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

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

Authors

Fuguo Jiang¹, David W Taylor², Janice S Chen¹, Jack E Kornfeld³, Kaihong Zhou³, Aubri J Thompson⁴, Eva Nogales⁵, Jennifer A Doudna⁶

Affiliations

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems*
Catalytic Domain
Clustered Regularly Interspaced Short Palindromic Repeats*
Crystallography, X-Ray
DNA / chemistry*
DNA Cleavage*
Endonucleases / chemistry*
Endonucleases / ultrastructure
Genetic Engineering
Genome
Nucleic Acid Conformation
Protein Conformation
RNA / chemistry
RNA, Guide, CRISPR-Cas Systems
Streptococcus pyogenes / enzymology*

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

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