Details

Autor(en) / Beteiligte

• Maji, Basudeb
• Gangopadhyay, Soumyashree A.
• Lee, Miseon
• Shi, Mengchao
• Wu, Peng
• Heler, Robert
• Mok, Beverly
• Lim, Donghyun
• Siriwardena, Sachini U.
• Paul, Bishwajit
• Dančík, Vlado
• Vetere, Amedeo
• Mesleh, Michael F.
• Marraffini, Luciano A.
• Liu, David R.
• Clemons, Paul A.
• Wagner, Bridget K.
• Choudhary, Amit

Titel

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

Ist Teil von

• Cell, 2019-05, Vol.177 (4), p.1067-1079.e19

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2019

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. [Display omitted] •Developed high-throughput assays for SpCas9 and performed a small-molecule screen•Identified reversible and cell-permeable inhibitors that disrupt SpCas9-DNA binding•Inhibitors allow dose and temporal control of (non)-nuclease-based SpCas9 systems•Identified a pharmacophore for SpCas9 inhibition using structure-activity studies A suite of high-throughput assays enables discovery of small-molecule inhibitors of CRISPR-Cas9 that are cell permeable, and non-toxic, providing a chemical means to control SpCas9-based tools.