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

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

Author information

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.

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