Fresh selection strain grown overnight was dispersed in 384-well selection plates before automated compound transfer. (B) Overall scheme of the primary screening in this study. Constitutively expressed GFP was measured as an indicator of cell growth. Small molecules with anti-CRISPR activity can inhibit cell death by chloramphenicol. Sp圜as9 and gRNA are expressed under inducible promoter and target cat locus conferring chloramphenicol resistance. Design scheme and workflow of cell-based HTS in this study. Measuring fluorescence increase using a microplate reader allows for the detection of Sp圜as9 inhibitory activity with our selection strain in a high-throughput fashion ( Figure 1B). To circumvent this, we additionally expressed green fluorescent protein (GFP) in our selection strain as a sensitive indicator of cell growth, which was measured in the primary screening process instead of optical density. Since many compounds may precipitate in an aqueous cell growth medium, measuring optical density to monitor cell growth could be problematic. (27) Here, we expected that HTS with our selection strain would also be applicable on small-molecule libraries. Previously, we employed our selection strain to functionally identify anti-CRISPRs from metagenomic libraries and showed in vivo activity of anti-CRISPRs. In the absence of a Sp圜as9 inhibitor, the cell will be susceptible to chloramphenicol, but with the inhibition of Sp圜as9, the cell becomes resistant to chloramphenicol. This complex targets DNA encoding chloramphenicol resistance marker cat, leading to direct coupling of chloramphenicol resistance and Sp圜as9 activity. (27) Sp圜as9 and gRNA are expressed from plasmids and form the Cas9:gRNA complex. coli selection strain for screening the Sp圜as9 inhibitory activity ( Figure 1A). Recently, one small-molecule Cas9 inhibitor was identified by fluorescence polarization-based high-throughput screening (HTS) (26) however, identifying new classes of Cas9 inhibitors with a different mechanism of action can also benefit and expand potential Cas9 applications in the future.įor cell-based HTS, we designed an E. Small molecules are relatively more permeable across the membrane, (23) proteolytically stable in vivo, (24) and generally non-immunogenic (25) compared to protein counterparts hence, they are more promising as tools or drugs for modulating the Cas9 activity. (21,22) Small molecules may be an attractive alternative to protein-based anti-CRISPR agents in some applications. (18) While protein-based anti-CRISPRs are useful tools to control the activity of Cas9, they have some limitations: (1) proteins are relatively large and thus cell membrane permeability is limited, (19,20) (2) proteins and small peptides tend to be quickly degraded in vivo by endogenous proteases, reducing their half-life and bioavailability, (20) and (3) proteins administered in vivo can potentially trigger undesired immune responses. Anti-CRISPR proteins were used to reduce off-targeting by Cas9 in human cells (8) and to suppress a gene drive in yeast. (15−17) By their natural properties, anti-CRISPR proteins have been used for optimizing CRISPR-based applications. (12−14) The genes encoding these proteins are commonly found in phages, plasmids, and mobile elements to counteract CRISPR/Cas-based immunity in the host cell. These molecules may find use as Cas9 modulators in various applications.Īnti-CRISPR proteins have been found in nature as natural defense mechanisms against CRISPR/Cas systems. 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. Extensive structure–activity relationship studies, which involved a deconstruction–reconstruction strategy, resulted in a range of analogues with significant improvements in the inhibitory activity. Here, we identified a small-molecule ligand with anti-CRISPR/Cas9 activity through a high-throughput screening utilizing an Escherichia coli selection system. Instead, small-molecule Cas9 inhibitors could serve as useful tools due to their permeable, proteolytically stable, and non-immunogenic nature. Anti-CRISPR proteins are known to inhibit the CRISPR/Cas adaptive immunity however, in vivo delivery of such proteins is problematic. CRISPR/Cas9 has revolutionized several areas of life science however, methods to control the Cas9 activity are needed for both scientific and therapeutic applications.