Rxivist logo

A Conformational Checkpoint Between DNA Binding And Cleavage By CRISPR-Cas9

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

Posted 30 Mar 2017
bioRxiv DOI: 10.1101/122242 (published DOI: 10.1126/sciadv.aao0027)

The Cas9 endonuclease is widely utilized 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 FRET, we identified an intermediate state of S. 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.

Download data

  • Downloaded 1,520 times
  • Download rankings, all-time:
    • Site-wide: 5,965 out of 92,351
    • In biophysics: 171 out of 4,011
  • Year to date:
    • Site-wide: 64,180 out of 92,351
  • Since beginning of last month:
    • Site-wide: 63,409 out of 92,351

Altmetric data


Downloads over time

Distribution of downloads per paper, site-wide


PanLingua

Sign up for the Rxivist weekly newsletter! (Click here for more details.)


News