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Anti-CRISPR Small Molecule Discovered Using New High-Throughput Screening Platform

NEW YORK (GenomeWeb) – A team led by researchers at the Broad Institute and Brigham and Women's Hospital has developed a high-throughput platform for identifying small molecules that can disrupt the genome editing activity of CRISPR-Cas nucleases.

The team developed a suite of high-throughput assays to measure SpCas9 functions, including an assay that screens for SpCas9 binding to the protospacer adjacent motif (PAM), and used them to screen a structurally diverse collection of natural-product-like small molecules. The ultimate goal was to identify compounds that disrupt the interaction between SpCas9 and DNA.

Indeed, as the researchers reported in a study in Cell today, their platform has identified the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9). The inhibitors weigh less than 500 Da and are cell permeable, reversible, and stable under physiological conditions, the researchers noted.

"Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships," they wrote.

SpCas9 inhibitors could be used for various purposes, according to the authors: to reduce mosaicism in germline editing; to temporarily switch off gene drives that propagate lethal traits; to inhibit SpCas9-mediated toxicity to helper cells in order to enable the efficient packaging of SpCas9 in adeno-associated viruses for delivery; to help allay concerns over dual use, where research that is designed to provide a benefit could be co-opted to do harm; to increase the research community's understanding of the biological functions of endogenous SpCas9; and to provide benefits to dCas9-based technologies, including base editing, from the perspectives of dose and temporal control.

In order to create their platform for the rapid identification and validation of small-molecule inhibitors of SpCas9, the researchers developed high-throughput assays for SpCas9 activity, including a fluorescence-polarization-based primary screening assay that probed the interaction of SpCas9 with PAM sequences. Using this primary screening assay, they sampled a set of small-molecule libraries derived from diversity-oriented synthesis to identify specific libraries enriched for screening hits.

Through a focused screen of the enriched libraries, they identified the molecule BRD0539 as a SpCas9 inhibitor. They then validated the activity of BRD0539 in multiple biochemical and cell-based assays and demonstrated target engagement by BRD0539 in cells. The team found that BRD0539 is stable in human plasma and reversibly inhibits SpCas9. Further, they noted that BRD0539 is cell permeable, unlike naturally occurring protein-based anti-CRISPRs, which require delivery methods like nucleofection.

The researchers then set out to determine BRD0539's specificity in its inhibition of editing activity. They looked to see whether the molecule disrupted the binding of the guide RNA to SpCas9 and found that the addition of BRD0539 did not perturb the SpCas9:gRNA interaction. Next, they examined whether BRD0539 disrupted the interactions of SpCas9 with DNA and found that the molecule did indeed block the formation of the DNA-bound state in a dose-dependent fashion.

Importantly, however, the researchers confirmed that while BRD0539 was able to inhibit SpCas9, it was unable to inhibit the editing activity of the FnCpf1 nuclease, which is structurally different from SpCas9. This finding further highlighted the molecule's specificity, they noted.

The researchers said their future work will involve the identification of inhibitors for next-generation CRISPR systems and understanding their mode of inhibition, as well as the application of such inhibitors.

"The timely and partial inhibition (about 50 percent) of SpCas9 reduced off-target editing for several genes, including a fivefold reduction for β-globin (HBB)-targeting gRNA that is of therapeutic interest for sickle cell disease," they wrote. "Partial inhibition of SpCas9 by BRD0539 together with its cellular permeability, reversibility, and plasma stability should afford a facile method for reducing the off-target activity of SpCas9. Together, our studies point to the utility of invocation of chemical biology-based approaches for genome editing and functional genomics studies using CRISPR-based systems."

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