|Publication Type||Journal Article|
|Year of Publication||2019|
|Authors||Maji, 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 Published||2019 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.