Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics.

Abstract

To investigate the kinetics of Cas9-mediated double strand break generation and repair in vivo, we developed two new tools. The first, chemically inducible Cas9 (ciCas9), is a rapidly-activated, single-component Cas9 variant engineered using a novel domain replacement strategy. ciCas9 can be activated in a matter of minutes, and the level of ciCas9 specificity and activity can be tuned. The second tool, DSB-ddPCR, is a droplet digital PCR-based assay for double strand breaks. DSB-ddPCR is the first assay to demonstrate time-resolved, highly quantitative and targeted measurement of DSBs. Combining these tools facilitated an unprecedented exploration of the kinetics of Cas9-mediated DNA cleavage and repair. We find that sgRNAs targeting different sites generally produce cleavage within minutes and repair within an hour or two. However, we observe distinct kinetic profiles, even for proximal sites, suggesting that target sequence and chromatin state modulate cleavage and repair kinetics.