Maji, B. et al. Cell 177, 1067–1079 (2019).

Precise and fast control of the CRISPR–Cas9 system is desirable for many applications. Maji et al. present the first small-molecule inhibitors of Cas9, cell-permeable and stable compounds of less than 500 Da. The researchers screened a library of 2,652 compounds in an in vitro assay based on changes in fluorescence polarization after binding of Cas9 to a fluorophore-labeled oligo containing 12 PAM sites. Two molecules showed a dose-dependent inhibitory effect that was confirmed in two cell-based assays. Cas9 was blocked in its ability to cleave the target, and dCas9 fused to transcriptional activators or base editors was similarly inhibited. The researchers then tested 641 structural analogs to the most potent inhibitor in a cell-based assay—they looked for an absence of cytotoxicity and dose-dependent inhibition, and found two analogs with increased potency. Exploring the mechanism of inhibition for one compound, they saw that it does not interfere with the Cas9–sgRNA interaction but blocks the binding of the complex to DNA. The authors propose that their high-throughput screening assay will enable the discovery of inhibitors for other Cas species.