Optimization and In Vivo Application of an Inducible Split-Cas9 System

T cells play a critical role in immune responses, differentiating into effector cells during acute infection. A subset of these effector cells transitions into memory cells upon antigen clearance, providing long-term immunity. However, in chronic conditions such as cancer, persistent antigen exposure leads to T cell exhaustion. Exhausted T cells exhibit reduced cytotoxicity, impaired cytokine production, and upregulated expression of multiple inhibitory receptors. This dysfunctional state significantly compromises anti-tumor immunity and contributes to poor patient outcomes.

CRISPR technologies offer a powerful means to altering T cell fate by modifying cell state and function through targeted genome editing. However, traditional CRISPR/Cas9 systems lack the temporal control necessary for studying dynamic processes like T cell exhaustion. To address this, we will employ a chemically inducible split-Cas9 system in which Cas9 is split into two individually inactive fragments that can be reassembled in the presence of rapamycin, enabling precise control over gene editing.

By utilizing this approach, we aim to identify and target negative regulators of T cell function. This will enable us to gain insights into the molecular mechanisms underlying T cell exhaustion and develop therapies to reverse this dysfunctional state, ultimately enhancing anti-tumor immunity.