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Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a programmable RNA-guided DNA endonuclease that has allowed the facile introduction of genomic alterations. Cas9-based technologies have transformed genome engineering and the interrogation of genomic functions, but methods to control such technologies across numerous dimensions—including dose, time, specificity, orthogonality remained the limiting factors in its therapeutic intervention. Our chemical-controlled system can deliver these requirements on demand. We have demonstrated how a combination of bioengineering method in CRISPR/Cas9 system with chemical agents can be useful in controlling complex biological events like gene-editing and transcriptional regulation. In a second approach, we have developed a high throughput small-molecule screening platform and identified inhibitors of SpCas9. 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. |