11/8/2023 0 Comments Reaconverter pro 7.3.60 torrent![]() ![]() 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.Īnti-CRISPR proteins have been found in nature as natural defense mechanisms against CRISPR/Cas systems. (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. (15−17) By their natural properties, anti-CRISPR proteins have been used for optimizing CRISPR-based applications. Anti-CRISPR proteins were used to reduce off-targeting by Cas9 in human cells (8) and to suppress a gene drive in yeast. (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. (21,22) Small molecules may be an attractive alternative to protein-based anti-CRISPR agents in some applications. 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. 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. coli selection strain for screening the Sp圜as9 inhibitory activity ( Figure 1A). (27) Sp圜as9 and gRNA are expressed from plasmids and form the Cas9:gRNA complex. This complex targets DNA encoding chloramphenicol resistance marker cat, leading to direct coupling of chloramphenicol resistance and Sp圜as9 activity. 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. Previously, we employed our selection strain to functionally identify anti-CRISPRs from metagenomic libraries and showed in vivo activity of anti-CRISPRs. (27) Here, we expected that HTS with our selection strain would also be applicable on small-molecule libraries. Since many compounds may precipitate in an aqueous cell growth medium, measuring optical density to monitor cell growth could be problematic. 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. 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). Design scheme and workflow of cell-based HTS in this study. Sp圜as9 and gRNA are expressed under inducible promoter and target cat locus conferring chloramphenicol resistance. Small molecules with anti-CRISPR activity can inhibit cell death by chloramphenicol. Constitutively expressed GFP was measured as an indicator of cell growth. (B) Overall scheme of the primary screening in this study. Fresh selection strain grown overnight was dispersed in 384-well selection plates before automated compound transfer. ![]()
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