CRISPR-Cas9 genome editing: Transfection of TnT cells and Jurkat-derived cell lines was performed by electroporation using the 4D Nucleofector device (Lonza) and the SE cell line kit (Lonza, #V4XC-1024). The day before transfection, cells were seeded at 2.5x10^5 cells/mL and cultured for 24 h. Prior to transfection, gRNA molecules (Suppl. Table 7) were assembled by mixing 4 µl of custom Alt-R crRNA (200 µM, IDT) with 4 µL of Alt-R tracrRNA (200 µM, IDT, #1072534), incubating the mix at 95°C for 5 min and cooling it to RT. For transfection of Cas9-negative cell lines, 2 µL of assembled gRNA molecules were mixed with 2 µL of recombinant SpCas9 (61 µM, IDT, #1081059) and incubated for > 10 min at room temperature to generate Cas9 RNP complexes. Immediately prior to transfection, cells were washed twice in PBS and 1x10^6 cells were re-suspended in 100 µL of SE buffer. 1.5 µg of HDR template and 7 µL of assembled gRNA (or 4 µL of Cas9 RNP complexes) were added to the cell suspension, mixed and transferred into a 1 mL electroporation cuvette. Cells were electroporated using program CK116, topped-up with 1 mL of complete media and rested for 10 min prior to transfer into a 12-well plate. Alt-R HDR enhancer (IDT, #1081073) was added at a 30 µM final concentration and removed after 16 h of culture by centrifugation. HDR efficiency was assessed by flow cytometry on day 5 post-transfection. For transfections at the 20 µL scale (Lonza, 557 #V4XC-1032), cell numbers and reagent volumes were reduced 5-fold.

Combinatorial TCRA3 library screening and selections: Combinatorial library HDR templates (20 µg) and CDR3B gRNA (10 nmol) were used to transfect 1x10^8 TnT cells using the 4D-Nucleofector LV unit (Lonza, #AAF-1002L). TnT cells with restored CD3 surface expression were bulk-sorted (SEL 1) on day 6 post transfection.

Primary T cell culture and genome editing: Human peripheral blood mononuclear cells were purchased from Stemcell Technologies (#70025) and CD8+ T cells isolated using the EasySep Human CD8+ T Cell Isolation kit (Stemcell Technologies, #17953). 720 Primary human CD8+ T cells were cultured for up to 24 days in ATCC-modified RPMI (Thermo Fisher, #A1049101) supplemented with 10% FBS, 10 mM non-essential amino acids, 50 µM 2-mercaptoethanol, 50 U ml-1 penicillin, 50 µg ml-1 streptomycin and freshly added 20 ng ml-1 recombinant human IL-2,(Peprotech, #200-02). T cells were activated with anti-CD3/anti-CD28 tetrameric antibody complexes (Stemcell Technologies, #10971) on days 1 and 13 of culture and expanded every 3-4 days. Transfection of primary T cells with Cas9 RNP complexes and TCRßa HDR templates was performed 3-4 days following activation using the 4D-Nucleofector and a 20 uL format P3 Primary Cell kit (Lonza, V4XP-3032). Briefly, 1x10^6 primary CD8+ T cells were transfected with 1 µg of HDR template, 1 µl of TRAC Cas9 RNP complex and 1 µl of TRBC1/2 Cas9 RNP complex using the EO-115 electroporation program (Cas9 RNP complexes= 50 µM gRNA, 30.5 µM recombinant SpCas9).