Schematic of the experimental workflow. Following identification of mutation(s) and before the experiment, cDNA fragments containing the mutation and the appropriate restriction enzyme cloning sites (supplemental Figure 1) are ordered from a commercial supplier and cloned into the WT cDNA in the MSCV-IRES-GFP backbone. Cloning, sequence verification, and plasmid amplification (maxi-prep) take 5 days. On day 0: naïve CD8+ T cells are isolated from a mouse spleen followed by nucleofection with Cas9 ribonucleoprotein to KO the desired genes. The KO cells are retrovirally transduced with recombinant human orthologues of the KO gene or empty MSCV-IRES-GFP vector as control and activated using BL/6 mouse T-cell depleted splenocyte “feeder cells,” simultaneously. On day 3 of cell expansion, the cells are sorted by flow cytometry with respect to their GFP reporter mean fluorescence intensity (identical for all samples on the day of sorting) to obtain pure populations expressing the desired protein. Experiments to test the function of the generated T cells can be conducted throughout days 4 to 7 and include 51Cr-release assays to test cytotoxicity, degranulation assays measuring LAMP-1/CD107a exposure to measure the exocytosis of cytotoxic granules, and western immunoblotting to determine the levels of protein expression. cDNA, complementary DNA; GFP, green fluorescent protein.
