Rheostatic control of Cas9-mediated DNA double strand break (DSB) generation and genome editing.

We recently reported two novel tools for precisely controlling and quantifying Cas9 activity: a chemically-inducible Cas9 variant (ciCas9) that can be rapidly activated by small molecules and a ddPCR assay for time-resolved measurement of DNA double strand breaks (DSB-ddPCR). Here, we further demonstrate the potential of ciCas9 to function as a tunable rheostat for Cas9 function. We show that a new, highly potent and selective small molecule activator paired with a more tightly regulated ciCas9 variant expands the range of accessible Cas9 activity levels. We subsequently demonstrate that ciCas9 activity levels can be dose-dependently tuned with a small molecule activator, facilitating rheostatic timecourse experiments. These studies provide the first insight into how Cas9-mediated DSB levels correlate with overall editing efficiency. Thus, we demonstrate that ciCas9 and our DSB-ddPCR assay permit the time-resolved study of Cas9 DSB generation and genome editing kinetics at a wide range of Cas9 activity levels.