New Platform Efficiently Identifies Small-Molecule Inhibitors of CRISPR-Cas9

By Will Boggs MD

June 12, 2019

NEW YORK (Reuters Health) - A new platform efficiently identifies small-molecule inhibitors that allow control of CRISPR-Cas9 activity in genome-engineering technologies, researchers report.

"Several applications of CRISPR enzymes (therapeutic or otherwise) can benefit from control of their activity over the dimensions of dose and time," said Dr. Amit Choudhary from Broad Institute of MIT and Harvard, in Cambridge, Massachusetts.

"In contrast to protein-based inhibitors for CRISPR systems found in viruses, the newly identified molecules are small, readily reversible, and potentially non-immunogenic, and they enter cells more efficiently," he told Reuters Health by email.

Streptococcus pyogenes Cas9 (SpCas9) is being developed as a gene therapy agent for multiple pathologies. Precise control of SpCas9 is critical, as elevated SpCas9 activity can be associated with off-target effects, chromosomal translocations, and genotoxicity. Ideally, its activity should be terminated immediately following on-target editing.

Dr. Choudhary and colleagues developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for its binding to the protospacer adjacent motif (PAM), and used these assays to screen a diverse collection of small molecules to identify compounds that disrupt the interaction between SpCas9 and DNA.

Based on the results from the primary screening assay that probed the SpCas9-PAM interaction, the researchers sampled a set of small-molecule libraries and identified BRD0539 as a specific SpCas9 inhibitor. BRD0539 is cell permeable, unlike protein-based anti-CRISPRs, and reversibly inhibits SpCas9.

Structure-activity studies showed that BRD0539 appears to inhibit SpCas9 by blocking the formation of the DNA-bound state rather than by interfering with the interaction between SpCas9 and guide RNA, the team reports in Cell, online May 2.

"A successful implementation of the screening platform and validation of its ability to identify inhibitors, and demonstration of a dose, spatial, and reversible control of Cas9 activity, opens the possibility of gene-editing more safely in a cellular context," Dr. Choudhary said. "However, the identified inhibitors employing the screening platform might require further optimization for their efficient activity in an organismal context. Also, the cellular off-targets of the identified inhibitors need to be investigated comprehensively."

"If our study is the foundation of a building, there are still lots of structure design, construction, and interior decorations to be done before the building can be open to the public - i.e., before the benefits of applying our small molecules will reach a general audience," he said.

Dr. Joseph Bondy-Denomy of the University of California, San Francisco, who recently reviewed protein inhibitors of CRISPR-Cas9, told Reuters Health by email, "I think this will provide an amazing research tool, and CRISPR is being used as a tool in nearly every lab in the world, to discovery new drug targets, gene pathways, etc. So this will really enable researchers to fine-tune CRISPR activity, which could help experiments."

"If/when CRISPR is in the clinic, being able to turn it off (with a drug or a protein) could really enhance safety," he said.

"These molecules will be very interesting reagents to probe Cas9 in the lab and will motivate our continued search for novel anti-CRISPR mechanisms in nature," said Dr. Bondy-Denomy, who was not involved in the research. "Perhaps there are small molecules that bacterial viruses make that turn off Cas9 in nature."


Cell 2019.