Choi, Yun-Seok

• Postdoctoral, Protein Engineering, Colorado State University, Fort Collins, CO 

• Postdoctoral, Structural Biology, Korea Basic Science Institute, South Korea 

• Ph.D., Bio-Analytical Science, University of Science & Technology, South Korea 

• M.S., Organic Chemistry, Hanyang University, South Korea 

• B.S., Chemistry, Hanyang University, South Korea

Title: Development of a Regulatory Switch to Control CRISPR/Cas9 Activity and Minimize Off-Target Effects

Research Summary: CRISPR/Cas9 genome editing has revolutionized both research and clinical applications, yet the prolonged presence of Cas9 in the nucleus can lead to off-target effects that compromise safety. This project proposes the development of a light-controlled turn- off switch for Cas9 that offers precise temporal regulation and irreversible deactivation of its activity. The approach involves incorporating a sortase-cleavable peptide into a non-disruptive loop of the Cas9 protein and co-expressing an engineered sortase enzyme. By fusing red light- responsive optogenetic domains to both the modified Cas9 and sortase, exposure to a specific wavelength of red light—known for minimizing DNA damage compared to shorter wavelengths— will induce heterodimerization, thereby facilitating sortase-mediated cleavage. This cleavage results in the irreversible removal of Cas9 from the system, ensuring that its activity is permanently halted once the desired genetic modifications are achieved. The experimental plan includes designing and characterizing several candidate constructs using structural modeling tools to validate in vitro activity, integrating the optogenetic system to assess light-induced cleavage efficiency and specificity, and employing comprehensive off-target analysis via Digenome-seq to confirm that the system minimizes unintended genomic modifications. Building on a strong track record of SD INBRE-supported research—including peer-reviewed publications, conference presentations, and successfully funded grants—this project leverages critical resources from the Research Match Cores for next-generation sequencing and high-throughput bioinformatics analysis. Ultimately, the expected outcome is a robust, user-controlled regulatory switch that not only enhances the safety and precision of CRISPR/Cas9 gene editing for clinical applications but also provides valuable training opportunities for undergraduate students in precision medicine and molecular biology.