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Cell DOI:10.1016/j.cell.2019.04.009

A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9.

Publication TypeJournal Article
Year of Publication2019
AuthorsMaji, B, Gangopadhyay, SA, Lee, M, Shi, M, Wu, P, Heler, R, Mok, B, Lim, D, Siriwardena, SU, Paul, B, Dančík, V, Vetere, A, Mesleh, MF, Marraffini, LA, Liu, DR, Clemons, PA, Wagner, BK, Choudhary, A
Date Published2019 May 02

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.


Alternate JournalCell
PubMed ID31051099