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

Abstract

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.

Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells

“…The structure of SP24 is different from previously reported small-molecule inhibitors [ 33 , 48 ], and the binding pocket is likely to be different as well. In fact, our data suggest that SpCas9 selectivity could be improved by the compounds.…”

Identification of a Cryptic Binding Site in CRISPR-Cas9 for Targeted Inhibition

“…In preparing this paper, we noticed that the same group identified another SpCas9 inhibitor (BRD7586) with improved potency through their developed high-throughput screening pipeline . Meanwhile, another group reported a new class of small-molecule SpCas9 inhibitors that were proposed to impede SpCas9-guide RNA interaction rather than that of SpCas9-DNA shown for BRD0539 . We anticipate that the current computational protocol can be implemented in future work to gain a more comprehensive understanding of the differential mechanisms by which these inhibitors act on SpCas9.…”

Regulating CRISPR/Cas9 Using Streptavidin‐Biotin Interactions†

“…To tackle these challenges, it is imperative to design and develop CRISPR/Cas9 systems with controllable activity, leading to the emergence of anti‐CRISPR technology. [ 11 ] The regulation of the CRISPR process can be achieved through the utilization of various anti‐CRISPR proteins (Acrs), [ 12‐13 ] small‐molecule inhibitors targeting Cas proteins, [ 14‐16 ] and non‐protein inhibitors (NPSs). [ 17 ] These approaches serve to enhance the precision and controllability of gene editing.…”