Journal
CELL
Volume 177, Issue 4, Pages 1067-+Publisher
CELL PRESS
DOI: 10.1016/j.cell.2019.04.009
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Funding
- Burroughs Wellcome Fund (Career Award at the Scientific Interface)
- DARPA [Brdi N66001-17-2-4055, HR0011-17-2-0049]
- NIH [R21AI126239, RM1HG009490, R35 GM118062]
- Army Research Office [W911NF1610586]
- U.S. Department of Defense (DOD) [W911NF1610586] Funding Source: U.S. Department of Defense (DOD)
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The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.
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