Cas9 is a programmable RNA-guided DNA endonuclease that allows facile induction of genomic alterations and the rapid spread of these alterations in a population via gene drives. Cas9-based technologies require multidimensional control over half-life, dosage, and spatiotemporal as off-target effects, chromosomal translocations, immunogenicity, and genotoxicity are observed at its elevated dose or prolonged activity. We have developed several Cas9 controllers and enhancers to elevate its technologies to the fullest potential, pivoting on applications in β-cell engineering. Activation: We fused Cas9 to destabilized domains that are largely unfolded and targeted, along with Cas9, for proteasomal degradation. Addition of a small molecule stabilizes these domains and rescues the fusion protein from degradation. Using this engineered Cas9 system, we rapidly, reversibly, and dose-dependently control both genome editing and transcription technologies of Cas9. (Nat. Chem. Biol. 2017; Angew. Chem. Int. Ed. 2019) and the rate of spread of engineered genes in insects (Cell Rep. 2020). Inhibition: We developed two platforms to identify small-molecule inhibitors--one that focuses on DNA-binding (Cell 2019) and another on DNA cutting (submitted). These platforms are scalable, cheap, and rapid, allowing us to screen small molecules to identify inhibitors. These molecules are the first examples of cell-permeable anti-CRISPRs that reversibly inhibit Cas9, and timely administration of these molecules enhanced Cas9 specificity by ~7-fold. Degradation: 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 (ACS Cent Sci 2020). This system was able to control the Cas9 activity in a specific and dose-dependent manner, and its use ameliorated the off-target effects of the enzyme and biased the DNA repair pathways. Enhancement: Precision genome editing involves coming together of four components, which dramatically reduces the editing efficiency. We developed a chemical conjugation platform that reduces the number of components to two and such chemically-enhanced Cas9 exhibited ~6-fold higher precision (Nat. Comm. 2020). We applied this Cas9 to engineer β-cells that secrete not only insulin but also immunoprotective agents (e.g., IL-10). We anticipate these systems to find widespread use in basic, biomedical, and defense research, as well as in therapeutic settings.

Chemical approaches to control and enhance CRISPR-based technologies

ACS Spring Meeting 2021

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