Extended Data Fig. 3: Direct comparison of common edited primary human T cell selection methods.

a, Intron knock-in of a tNGFR-CAR-PuroR synthetic exon enabled a single population of edited cells to be compatible with four common selection methods. Representative flow cytometric plots of TRAC intron CAR T cells after purification with four distinct selection methods. Knock-in of a synthetic exon containing a tNGFR-CAR-PuroR multicistronic cassette to TRAC intron 1 enabled successfully edited cells to be negatively selected by CD3 Depletion (removal of TCR positive cells), positively selected by streptavidin magnetic bead enrichment after binding of anti-tNGFR biotinylated antibodies, fluorescence-activated cell sorting after binding of anti-tNGFR fluorescent antibodies, or drug selection after culture in puromycin. Negative selection by CD3 depletion yields predominantly a successfully edited CAR + T cell population without the endogenous TCR, although rarer TCR negative / knock-in negative cells are present (likely due to the RNP induced double stranded break within the intron causing a large deletion that included a portion of one or both adjacent exons, instead of the more common NHEJ repair outcome of smaller indels). Positive selection, sorting, and drug selection in contrast remove all knock-in negative cells, but retain a population of TCR positive / knock-in positive cells (likely due to successful HDR mediated knock-in to one TRAC allele with either no editing or a small indel removed during mRNA splicing on the second TRAC allele; while in some T cells one TCRα loci is silenced, numerous T cells express functional TCRα chains from both alleles⁴⁸). Only negative selections do not require additional genetic material (for example no selection marker or resistance gene necessary) and leave cells untouched post-selection. b, Percentage of residual TCR positive cells following four different selection methods. TRAC intron knock-in followed by CD3 Negative Selection (Blue) leaves almost no detectable TCR+ cells remaining. c, In vitro killing of Nalm6 target cells by TRAC intron knock-in CD19-28z CAR T cells following negative, positive, sorting, or drug selection, measured 48 hours post co-incubation. n = 4 unique donors. ns = not significant, * = P < 0.05, Two-sided paired t test. d, In vitro proliferation of TRAC intron knock-in CD19-28z CAR T cells following negative, positive, sorting, or drug selection after co-culture with Nalm6 target cells. Error bars represent standard error of the mean from n = 4 unique donors. ns = not significant, * = P < 0.05, Two-sided Mann-Whitney test. e, Puromycin dose titrations reveal a tradeoff between purity and cell yield when performing drug selections. Increasing doses of puromycin yielded greater T cell purity, but overall edited cell yield began to decline with increasing puromycin concentrations. Beginning 6 days post editing, 100,000 bulk edited T cells were treated with indicated doses of puromycin for 48 hours. A dose of 5 ug/mL was used for experiments in Fig. 2b-c to balance purity and yield. n = 2 donors with 4 technical replicates each.