Abstract：
: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 time-course 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.
journal_name
ACS Chem Bioljournal_title
ACS chemical biologyauthors
Rose JC,Stephany JJ,Wei CT,Fowler DM,Maly DJdoi
10.1021/acschembio.7b00652subject
Has Abstractpub_date
2018-02-16 00:00:00pages
438-442issue
2eissn
1554-8929issn
1554-8937journal_volume
13pub_type
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