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

Maji B, Gangopadhyay SA, Lee M, Shi M, Wu P, Heler R, Mok B, Lim D, Siriwardena SU, Paul B, Dancík V, Vetere A, Mesleh MF, Marraffini LA, Liu DR, Clemons PA, Wagner BK, Choudhary A
Source: Cell Res
Publication Date: (2019)
Issue: 177(4): 1067-1079
Research Area:
Gene Expression
Basic Research
Cells used in publication:
U-2 OS
Species: human
Tissue Origin: bone
4D-Nucleofector® X-Unit

SpCas9 nuclease activity in eGFP-disruption assay. 

Approximately 200,000 U2OS.eGFP-PEST cells were nucleofected following two different methods: either by nucleofecting plasmids or by ribonucleoprotein (RNP) using the SE Cell Line 4D-Nucleofector™ X Kit (Lonza) according to the manufacturer’s protocol. In the plasmid nucleofection method, cells were nucleofected with either 440 ng of SpCas9 (Addgene Plasmid #43861) or 400 ng of SpCas9 with 40 ng of gRNA (pFYF1320 eGFP Site#1, Addgene Plasmid #47511). Cells nucleofected only with SpCas9-expressing plasmid were used as the transfection control. In the RNP-nucleofection method, either 20 mmol of SpCas9 or 20 pmol of preformed SpCas9:gRNA (eGFP Site#1) complex were nucleofected. Approximately 20,000 transfected cells/well were plated in four replicates in a 96-well plate (Corning® 3904).


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.