2. Academic Validation
  3. A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9

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

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: achoudhary@bwh.harvard.edu.
PMID: 31051099 DOI: 10.1016/j.cell.2019.04.009
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.

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