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 Light Controlled Turn Off Switch for CRISPR Cas9 to Minimize Off Target Effects

The CRISPR Cas9 system is a powerful tool for gene editing but its prolonged presence in cells can lead to unintended DNA edits that may compromise safety. Our research at Black Hills State University aims to solve this by creating a light sensitive switch. By using protein engineering we are developing a system that allows researchers to deactivate Cas9 activity at a precise moment using specific wavelengths of light. This ensures the enzyme only works long enough to complete the intended edit.

Our approach involved dual component system consisting of a modified Cas9 protein and a specialized regulatory enzyme. We have engineered a specific cleavage site into a flexible region of the Cas9 structure. Both the Cas9 and the regulatory enzyme are fused to optogenetic domains that act as light dependent connectors.

When triggered by light these components join together which allows the enzyme to cleave and neutralize the Cas9 protein. This system is designed with specific kinetic properties to ensure that the switch remains stable in the dark and only activates under controlled light simulation. This provides a level of temporal control that is currently missing from standard CRISPR applications.

Supported by SD INBRE and an NIH R15 grant we have already successfully modeled adn validated the initial protein constructs. Our current work focuses on measuring the efficiency of this light triggered deactivation and using high resolution genomic sequencing to confirm the reduction in unintended activity.