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

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.