Primary Human T Cell Isolation and Culture
PBMCs from healthy human blood donors were collected under an approved IRB protocol by the Stanford Blood Center and used to isolate human T cells. Briefly, leukoreduction chambers (LRS) from processing of platelet donations were used to isolate PBMCs using density centrifugation with Ficoll (Lymphoprep, StemCell) within SepMate tubes (StemCell) according to manufacturer’s instructions. Next, primary human CD3 positive T cells were isolated by negative selection using Human CD3 T Cell Enrichment kit (StemCell) according to manufacturer’s instructions. Isolated primary human CD3 T cells were counted using an automated cell counter (Countess, Thermo), and activated using anti-human CD3/CD28 dynabeads (Cell Therapy Systems, Thermo) at a 1:1 ratio in XVivo 15 media (Lonza) supplemented with 5% FBS (MilliporeSigma) and 50 U/mL of human IL-2 (Peprotech). T cells were activated at 1:1 ratio of cells to dynabeads, and initially cultured in standard tissue culture incubators at approximately 1e6 cells / mL media. After gene editing/electroporations, T cells were counted and reseeded at approximately 1e6 cells / mL XVivo 15 media with fresh IL-2 every 2-3 days.

Non-viral gene knockins
Two days after activation, human T cells were harvested, dynabeads were magnetically removed by incubating for two minutes at room temperature on a magnet (EasySep Magnet, StemCell), and cells were counted using an automated cytometer. For electroporations, one million T cells per editing condition were gently pelleted by centrifugation at 90G for 10 minutes, followed by careful aspiration of the supernatant. T cell pellets were resuspended in 20 µL per editing condition in P3 Buffer (Lonza) and then mixed with prepared RNP and DNA HDRT templates. For each Cas9 knockin condition, RNPs were prepared by first complexing the gRNA by mixing 0.375 uL of 200 µM tracrRNA (IDT) with 0.375 uL of 200 uM crRNA (IDT) and incubating for 15 minutes at room temperature. Next 0.25 uL of 100 mg/mL PGA (15–50 kDa poly(L-glutamic acid); MilliporeSigma) was then added to the complexed gRNA and mixed by pipetting up and down. Next 0.5 µL of 40 µM SpCas9 (UC Berkeley MacroLab) was then added, mixed by pipetting up and down, and incubated for 15 minutes at room temperature to form the final Cas9 RNP. For Cas9 knockins, 20 µL of T cells were mixed with 1.5 µL of RNP (20 pmols total RNP) and 4 µL of plasmid DNA HDR Template at 1 µg/µL (4 ugs total HDRT). For each Cas12a condition, RNPs were prepared by first mixing 0.4 µL of 200 µM Cas12a gRNA (IDT) with 0.2 µL of 100 mg/mL PGA and pipetting up and down. Next 0.4 µL of 60 uM AsUltraCas12a (UC Berkeley MacroLab) was added and mixed by pipetting up and down, followed by incubation at room temperature for 10 minutes. For Cas12 a knockins, 20 µL of T cells were mixed with 1 µL of Cas12a RNP (24 pmols total RNP) and 4 uL of plasmid DNA HDR Template at 1 ug/uL (4 ugs total HDRT). For both Cas9 and Cas12a knockins, T cells were electropoated on a Gen2 Lonza 4D electroporation/nucleofection system using 96 well plate attachment and 20 µL cuvettes, using pulse code EO-151. Immediately following electroporation, 75 µL of pre-warmed XVivo 15 media was added to each cuvette, and cells were rested within the cuvettes for 15 minutes in a standard 37°C Tissue Culture Incubator prior to moving to culture plates or flasks. An annotated list of all gRNA sequences used in the study is available in Supplementary Table 1.