A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

Yavuz S Dagdas; Janice S Chen; Samuel H Sternberg; Jennifer A Doudna; Ahmet Yildiz

doi:10.1126/sciadv.aao0027

A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

Sci Adv. 2017 Aug 4;3(8):eaao0027. doi: 10.1126/sciadv.aao0027. eCollection 2017 Aug.

Authors

Yavuz S Dagdas¹, Janice S Chen², Samuel H Sternberg³, Jennifer A Doudna^{2

3

4

5}, Ahmet Yildiz^{2

6}

Affiliations

¹ Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
⁶ Department of Physics, University of California, Berkeley, Berkeley, CA 94720, USA.

Abstract

The Cas9 endonuclease is widely used for genome engineering applications by programming its single-guide RNA, and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer, we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Cas Systems*
DNA / chemistry*
DNA / metabolism*
DNA Cleavage*
Fluorescence Resonance Energy Transfer
Gene Editing
Molecular Conformation
Molecular Dynamics Simulation
Protein Domains
RNA, Guide, CRISPR-Cas Systems
Structure-Activity Relationship

Substances

RNA, Guide, CRISPR-Cas Systems
DNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding