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

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

doi:10.1126/sciadv.aao0027

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

Journal Article · Tue Aug 01 00:00:00 EDT 2017 · Science Advances

DOI:https://doi.org/10.1126/sciadv.aao0027· OSTI ID:1625977

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Univ. of California, Berkeley, CA (United States). Biophysics Graduate Group; DOE/OSTI
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Physical Biosciences Division
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). Dept. of Physics

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1625977

Journal Information:: Science Advances, Journal Name: Science Advances Journal Issue: 8 Vol. 3; ISSN 2375-2548

Publisher:: AAASCopyright Statement

Country of Publication:: United States

Language:: English

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