Fig 1: bioPROTACs can be composed into circuits for cell autonomous modulation of CAR T cell signaling. (A) CRISPR/Cas9 knock-in methods can be used to guide bioPROTACs to endogenous proteins of interest. (B) Genetic circuit that uses SNIPR antigen detection to trigger bioPROTAC expression resulting in GFP-ZAP70 degradation. (C) SNIPR-activated bioPROTACs induced degradation of GFP-ZAP70 as measured by GFP fluorescence. Engineered T cells were challenged with K562 target cells expressing CD19 or CD19 and HER2 at an E:T ratio of 1:1 for 48 h. GFP fluorescence was measured by flow cytometry. Dots represent technical replicates, and error shows SEM. (D) Multiple challenge assay used to assess circuit functionality. (E) T cell proliferation and survival is affected by bioPROTAC-mediated knockdown of GFP-ZAP70. T cell numbers were quantified using flow cytometry and normalized to the no degron control for each antigen condition. T cell counts were normalized to the no degron control and then again normalized to the first challenge. Dots are the mean of three technical replicates, and error bars represent SEM. (F) bioPROTAC-mediated knockdown of GFP-ZAP70 resulted in a decrease in CD25 expression. Dots represent technical replicates. The error shown represents SEM. CD25 fluorescence values were normalized to those of cells expressing the no degron control circuit. (G) SNIPR-induced bioPROTACs are capable of inducing GFP-ZAP70 degradation as measured by total ZAP70 levels. ZAP70 fluorescence was measured by intracellular staining followed by flow cytometry. ZAP70+ cells were determined by in silico gating relative to the unstained control. Each dot represents technical replicates, and error bars show SEM. An unpaired t test was used for all statistical comparisons. *P < 0.01.