Journal article
Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Technical University of Denmark2
DTU Microbes Initiative, Centers, Technical University of Denmark3
Bacterial Synthetic Biology, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
Center for Microbial Secondary Metabolites, Centers, Technical University of Denmark5
Department of Chemistry, Technical University of Denmark6
University of Copenhagen7
CRISPR/Cas9 has revolutionized several areas of life science; however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity; however, in vivo delivery of such proteins is problematic.
Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system.
Extensive structure-activity relationship studies, which involved a deconstruction-reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Based on NMR and electrophoretic mobility shift assays, we propose that the inhibitory action of these compounds likely results from direct binding to apo-Cas9, preventing Cas9:gRNA complex formation.
These molecules may find use as Cas9 modulators in various applications.
Language: | English |
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Publisher: | American Chemical Society |
Year: | 2022 |
Pages: | 3266-3305 |
ISSN: | 15204804 and 00222623 |
Types: | Journal article |
DOI: | 10.1021/acs.jmedchem.1c01834 |
ORCIDs: | Lee, Sang-Woo , Vazquez-Uribe, Ruben , Gotfredsen, Charlotte Held , Clausen, Mads Hartvig , Sommer, Morten Otto Alexander , 0000-0002-8269-3941 and 0000-0003-4305-9910 |