Authors: Basudeb Maji, Soumyashree A. Gangopadhyay, Miseon Lee, Mengchao Shi, Peng Wu, Robert Heler, Beverly Mok, Donghyun Lim, Sachini U. Siriwardena, Bishwajit Paul, Vlado Dančík, Amedeo Vetere, Michael F. Mesleh, Luciano A. Marraffini, David R. Liu, Paul A. Clemons, Bridget K. Wagner, Amit Choudhary
Summary: 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.
Source: Cell, 2019; 177 (4): 1067