A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9 PMID: 31051099

ADC Targets in Oncology

2026-04-10

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

Cell. 2019 May 2;177(4):1067-1079.e19. doi: 10.1016/j.cell.2019.04.009.

Basudeb Maji ¹ , Soumyashree A Gangopadhyay ¹ , Miseon Lee ² , Mengchao Shi ¹ , Peng Wu ¹ , Robert Heler ³ , Beverly Mok ⁴ , Donghyun Lim ² , Sachini U Siriwardena ⁵ , Bishwajit Paul ¹ , Vlado Dančík ⁵ , Amedeo Vetere ⁵ , Michael F Mesleh ⁶ , Luciano A Marraffini ⁷ , David R Liu ⁸ , Paul A Clemons ⁵ , Bridget K Wagner ⁵ , Amit Choudhary ⁹

Affiliations

1. Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA.
2. Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
3. Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA.
4. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
5. Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
6. Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
7. Laboratory of Bacteriology, The Rockefeller University, New York, NY 10065, USA; Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 11231, USA.
8. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
9. Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA 02115, USA. Electronic address: [email protected].

Abstract

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.

Keywords

CRISPR-Cas9; S. pyogenes Cas9; base editing; dCas9; genome editing; high-throughput screening; small-molecule inhibitor; synthetic anti-CRISPRs; transcription activation.