Extended Data Fig. 1: Optimization of a CRISPR-HDR protocol for engineering primary murine T cells.
From: Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery
a. Schematic of CRISPR-HDR protocol using a PD-1/GFP dsDNA repair template. Cas9 and PD-1-targeting sgRNA RNPs were electroporated into activated murine T cells with a purified PD-1/GFP dsDNA repair template and stimulated 72 h later with plate-bound anti-CD3 and CD28 antibodies for 24 h before analysis of GFP by flow cytometry. b. Flow cytometry plots showing GFP expression in stimulated Mock, PD-1 KO or PD-1/GFP murine T cells, representative of n = 3 experiments. c. Schematic of CRISPR-HDR protocol using a PD-1/GFP AAV6 repair template. Cas9 and PD-1-targeting sgRNA RNPs were electroporated into activated murine T cells then incubated with a PD-1/GFP AAV6 repair template for 4 h prior to GFP analyses 72 h later as per (a). d. Flow cytometry plots (left) and quantification (right) of GFP expression in Mock, PD-1 KO or PD-1/GFP murine T cells edited with AAV6 at the indicated MOIs. Data represent mean ± SD of technical duplicates, representative of n = 2 experiments. e. Quantification of GFP expression in stimulated PD-1/GFP murine T cells edited with AAV6 at an MOI of 100 K, incubated at decreasing volumes to increase the effective AAV6 concentration, represented as mean ± SD of technical duplicates. f. Quantification of GFP expression in stimulated PD-1/GFP murine T cells edited with AAV6 at an MOI of 100 K and M3814 at the indicated concentrations, representative of n = 2 experiments. Illustrations in a and c created using BioRender: a, Chen, A., https://BioRender.com/ye4jk15 (2025); c, Chen, A., https://BioRender.com/ye4jk15 (2025).
