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

Basudeb Maji; Soumyashree A Gangopadhyay; Miseon Lee; Mengchao Shi; Peng Wu; Robert Heler; Beverly Mok; Donghyun Lim; Sachini U Siriwardena; Bishwajit Paul; Vlado Dančík; Amedeo Vetere; Michael F Mesleh; Luciano A Marraffini; David R Liu; Paul A Clemons; Bridget K Wagner; Amit Choudhary

doi:10.1016/j.cell.2019.04.009

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.

Authors

Basudeb Maji¹, Soumyashree A Gangopadhyay¹, Miseon Lee², Mengchao Shi¹, Peng Wu¹, Robert Heler³, Beverly Mok⁴, Donghyun Lim², Sachini U Siriwardena⁵, Bishwajit Paul¹, Vlado Dančík⁵, Amedeo Vetere⁵, Michael F Mesleh⁶, Luciano A Marraffini⁷, David R Liu⁸, Paul A Clemons⁵, Bridget K Wagner⁵, Amit Choudhary⁹

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.

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.

Published by Elsevier Inc.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Associated Protein 9 / antagonists & inhibitors*
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems / physiology*
Clustered Regularly Interspaced Short Palindromic Repeats / physiology
DNA / metabolism
Endonucleases / metabolism
Gene Editing / methods
Genome
High-Throughput Screening Assays / methods*
Small Molecule Libraries
Streptococcus pyogenes / genetics
Substrate Specificity

Substances

Small Molecule Libraries
DNA
CRISPR-Associated Protein 9
Cas9 endonuclease Streptococcus pyogenes
Endonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding