A critical need exists for a set of small molecules that activate, inhibit, or degrade SpCas9 as these molecules will allow precise regulation of SpCas9 activities across multiple dimensions, including dose, time, specificity, genotoxicity, and immunogenicity. Finely-tuned control of SpCas9 levels is important as high SpCas9 activity results in elevated off-targets and p53-mediated genotoxicity. Multidimensional control of SpCas9-based gene drives will be important from application and biosafety perspective. SpCas9 inhibitors, by disrupting CRISPR-immunity, will propel bacteria to evolve new CRISPR-systems. Rapid degradation of SpCas9 will allow evasion of the immune surveillance of the host and genotoxicity. Activators: We have previously reported small-molecule activators of SpCas9 in mammalian cells (Nat Chem Biol 2017). We re-engineered this system to work in insects that have a lower body temperature (25 deg C). In a typical gene drive inheritance, the percentage of super-Mendelian inheritance is binary—0 % or 100%. Using our system, we can precisely control the degree of super-Mendelian inheritance to 0%, 100% or any intermediate value by adjusting the amount of the small molecule in the insect’s food. Inhibitors: We have developed several high throughput SpCas9 assays for PAM-binding and using these assays we have identified small-molecule inhibitors that block SpCas9 nuclease activity, base-editing, and transcription activation in mammalian cells, and CRISPR-immunity in bacteria. Further, by targeting the nuclease domains of SpCas9, SaCas9, and Cpf1, we have identified broad-spectrum small-molecule inhibitors of these nucleases. Degraders: We have developed chimeric small-molecules that can bring SpCas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of SpCas9 by the proteasome. In addition to inhibiting the nuclease activity, these small-molecules enable precise temporal control of the half-life of SpCas9 allowing one to control the degree of immune response and genotoxicity. We anticipate our synthetic activators, inhibitors, and degraders to find widespread use in basic, biomedical, and defense research, as well as in therapeutic settings.

Synthetic activators, inhibitors, and degraders of CRISPR-associated nucleases

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.