4 years ago

Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics

Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics
Bridget M Trevillian, Dustin J Maly, Douglas M Fowler, John C Rose, Jason H Bielas, William J Valente, Jason J Stephany, Ha V Dang
We developed a chemically inducible Cas9 (ciCas9) and a droplet digital PCR assay for double-strand breaks (DSB-ddPCR) to investigate the kinetics of Cas9-mediated generation and repair of DSBs in cells. ciCas9 is a rapidly activated, single-component Cas9 variant engineered by replacing the protein's REC2 domain with the BCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCL-xL–BH3 interaction within minutes. DSB-ddPCR demonstrates 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 induce cleavage within minutes and repair within 1 or 2 h. However, we observe distinct kinetic profiles, even for proximal sites, and this suggests that target sequence and chromatin state modulate cleavage and repair kinetics.

Publisher URL: http://dx.doi.org/10.1038/nmeth.4368

DOI: 10.1038/nmeth.4368

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