CAREER: Develop PROTAC-CID, a Novel Small Molecule Inducible Platform for Controllable Gene Expression in Mammalian Cells

It is difficult to control gene expression in mammalian cells. Accomplishing this is key to developing effective cell therapies. Small molecules can exert that control. Unfortunately, small molecules are often toxic, and tend to have a limited, on-off capability. The aim of this project is to develop an improved small molecule system for mammalian cells. This system will be tunable instead of on/off. Highly sensitive, it will interact with multiple genes to direct more complex behaviors. Undergraduates from underrepresented groups and high school teachers will learn techniques of synthetic biology and CRISPR technology. Proteolysis Targeting Chimeric (PROTAC)-Chemically Induced Dimerization (CID) systems will be developed as sensitive, multiplex, tunable, and safe toolkits to control gene expression in mammalian cells. Combining PROTAC-CID with genome editors will enable precise genomic modification. This will greatly increase the accuracy and safety of using genome editors for basic and therapeutic applications. The specific research goals are to 1) establish a novel PROTAC-CID inducible gene expression system; 2) engineer multiplex inducible gene regulations; 3) design high-induction and low-basal level PROTAC-CID systems for Cre recombinase expression, and 4) develop PROTAC-CID inducible CRISPR base editors. Successful completion should result in novel PROTAC-CID inducible gene expression platforms for multiplex gene regulations in living eukaryotic cells. These systems will provide deep insights into the approaches to achieve high-induction and low basal-level gene expressions. Finally, applying PROTAC-CID systems to CRISPR base editors will permit inducible genomic DNA modification at a defined time to reduce the off-target effect and increase the accuracy and safety in genome editing. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.