A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules

Donghyun Lim; Qingxuan Zhou; Kurt J Cox; Benjamin K Law; Miseon Lee; Praveen Kokkonda; Vedagopuram Sreekanth; Rajaiah Pergu; Santosh K Chaudhary; Soumyashree A Gangopadhyay; Basudeb Maji; Sophia Lai; Yuka Amako; David B Thompson; Hari K K Subramanian; Michael F Mesleh; Vlado Dančík; Paul A Clemons; Bridget K Wagner; Christina M Woo; George M Church; Amit Choudhary

doi:10.1038/s41556-022-01005-8

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules

Nat Cell Biol. 2022 Dec;24(12):1766-1775. doi: 10.1038/s41556-022-01005-8. Epub 2022 Nov 17.

Authors

Donghyun Lim^#^{1

2

3}, Qingxuan Zhou^#^{1

2}, Kurt J Cox^#^{1

2}, Benjamin K Law^#^{1

2}, Miseon Lee^#^{1

2}, Praveen Kokkonda^#^{1

2}, Vedagopuram Sreekanth^#^{1

2

4}, Rajaiah Pergu^{1

2}, Santosh K Chaudhary^{1

2}, Soumyashree A Gangopadhyay^{1

2

4}, Basudeb Maji^{1

2

4}, Sophia Lai^{1

5}, Yuka Amako⁵, David B Thompson^{6

7}, Hari K K Subramanian⁸, Michael F Mesleh⁹, Vlado Dančík¹, Paul A Clemons¹, Bridget K Wagner¹, Christina M Woo⁵, George M Church^{6

7}, Amit Choudhary^{10

11

12}

Affiliations

¹ Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
² Department of Medicine, Harvard Medical School, Boston, MA, USA.
³ School of Biopharmaceutical and Medical Sciences, Sungshin University, Seoul, South Korea.
⁴ Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA, USA.
⁵ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
⁶ Department of Genetics, Harvard Medical School, Boston, MA, USA.
⁷ Wyss Institute for Biologically Inspired Engineering, Boston, MA, USA.
⁸ Department of Mechanical Engineering, University of California-Riverside, Riverside, CA, USA.
⁹ Center for the Development of Therapeutics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹⁰ Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard, Cambridge, MA, USA. achoudhary@bwh.harvard.edu.
¹¹ Department of Medicine, Harvard Medical School, Boston, MA, USA. achoudhary@bwh.harvard.edu.
¹² Divisions of Renal Medicine and Engineering, Brigham and Women's Hospital, Boston, MA, USA. achoudhary@bwh.harvard.edu.

^# Contributed equally.

Abstract

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules

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