Project Summary The complexity and dynamics of protein kinases and epigenetic modifications in cellular processes necessitate the development of precise biosensors for live imaging. This proposal aims to develop single-FP-based, high- performance, ultrasensitive biosensors through directed evolution in mammalian cells. These biosensors will be used in multiplexed imaging and dynamic visualization of signaling activities in situ, with a specific focus on improving chimeric antigen receptor T cells (CAR-T) for cancer immunotherapy. Addressing the limitations of CAR-T therapy, particularly T cell exhaustion in solid tumors, requires a better understanding of the molecular mechanisms involved. Although kinases and epigenetic markers, particularly H3K27me3, play key roles in T cell regulation, our understanding of their spatiotemporal dynamics during cancer-immune interactions and through the course of CAR-T cell rejuvenation remains limited due to the absence of appropriate investigative tools. Thus, parallel examination of these key regulators in cancer-immune interacting environments should reveal new insights into the systematic behaviors and identify essential links for therapeutic manipulation. My hypothesis is that 1) the reversal of CAR-T cell exhaustion involves a rejuvenation of ZAP70 and Lck kinase’s function and a reprogramming of the H3K27me3, transitioning from an exhausted state to a naïve T cell-like state; and 2) transient knockdown of exhaustion-related genes could prevent and reverse exhaustion of CAR-T cells, which can be reflected by the kinase and epigenetics coordinated response patterns. Leveraging the combined expertise of my mentors, I have demonstrated the feasibility of engineering single-FP biosensors for tyrosine kinases, directed evolution of single-FP biosensors in mammalian cells and established a transient gene knockdown system that can be remotely controlled by focused ultrasound (FUS). Building on these achievements, three distinct aims have been further proposed: Aim 1 focuses on engineering novel single-FP prototype biosensors for monitoring tyrosine kinases or epigenetics in CAR-T cells of different phenotypes during cancer cell engagement and establishing a multiplexed imaging platform. Aim 2 involves developing ultrasensitive single-FP biosensors through directed evolution, high-throughput screening, and next- generation sequencing. Aim 3 focuses on the application of these biosensors for multiplexed imaging and manipulation of kinase-epigenome signaling in CAR-T cells using a FUS controllable gene knockdown system. This biosensor engineering platform can potentially be extended to develop any other kinase and epigenetic biosensors for live cell imaging. Similarly, the novel FUS-controllable gene knockdown system could be generalized for broader manipulations of various cellular processes. Successful execution of this project could revolutionize biosensor engineering and kinase imaging, profoundly impacting o...