期刊
NATURE METHODS
卷 14, 期 9, 页码 891-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.4368
关键词
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资金
- NIH [R01GM086858, R01GM109110, F30CA189793]
- NSF [0954242]
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [0954242] Funding Source: National Science Foundation
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 BCLCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCLCL-xL-BH3 interaction within minutes. DSB-ddPCR demonstrates time-resolved, highly quantitative, and targeted measurement of DSDSBs. 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.
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