Knockout generation in resting human CD4+ T cells. Freshly isolated CD4+ T cells (2 × 106) were washed twice with PBS and resuspended in 20 µl buffer P3 (Lonza; V4XP-3032). In parallel, synthetic sgRNAs (Synthego) were incubated together with recombinant NLS-Cas9 (IDT; 1081059) for 10 min at room temperature, at a ratio of 1:2.5 (40 pmol Cas9 protein per 100 pmol gRNA) to form the CRISPR–CAS9–gRNA RNP complex. The CAS9–gRNA mix was diluted with sterile filtered (0.22 µm) PBS to reach a final concentration of 20 µM RNPs. For single gRNA editing, 5 µl of the 20 µM RNPs were then mixed with the cell suspension and transferred into a 16-well reaction cuvette of the 4D-Nucleofector System (Lonza). For efficient KO of individual targets, a mix of two specific, pre-validated gRNAs was used, if not indicated differently. Here, only 2 µl of RNP complexes for each gRNAs were used. For co-editing of up to six genes, only 0.5 µl of each RNP complex were used. Cells were nucleofected using program EH-100 on the 4D-Nucleofector system12. Then, 100 µl of pre-warmed RPMI (without supplements) was added to each well and cells were transferred to 48-well plates and allowed to recover for 10 min at 37 °C. Subsequently, complete culture medium
supplemented with IL-7 (Peprotech; 200-07) and IL-15 (Peprotech; 200-15) (2 ng ml-1 each) was added.

Knock in of resting CD4+ T cells. For KIs, the same nucleofection conditions as for the KO generation were used (P3 buffer and program EH-100; Lonza). In addition to the RNP, the donor DNA template was added to the P3 cell suspension at 1 µg, unless stated otherwise. Additional information on the overall strategies to generate KIs into different loci in resting CD4+ T cells, including DNA templates, gRNAs and primers is included in Extended Data Table 1.