Identification and Optimization of Novel Small-Molecule Cas9 Inhibitors by Cell-Based High-Throughput Screening

Sang-Woo Lee, Kim Tai Tran, Ruben Vazquez-Uribe, Charlotte Held Gotfredsen, Mads Hartvig Clausen, Blanca Lopez Mendez, Guillermo Montoya, Anders Bach*, Morten Otto Alexander Sommer*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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.
Original languageEnglish
JournalJournal of Medicinal Chemistry
Issue number4
Pages (from-to)3266–3305
Number of pages40
Publication statusPublished - 2022


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