Fig. 1: SEEDs couple transgene integration to the target protein disruption.
From: SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci
a, Overview of editing outcomes generated with an intron-targeted single gRNA (sgRNA) and a SEED HDRT. Immunomagnetic reagents are used to deplete cells that retain expression of the surface protein targeted by a SEED, thereby enriching for cells with transgene integration. b, Diagram of a TRAC intron-targeted SEED HDRT encoding a CAR and EGFRt. c,d, T cells were edited with TRAC intron-targeted RNP and HDRT (b) and then immunomagnetically purified with anti-TCR (n = 3 donors). c, Flow cytometry plots of TCR and CAR expression (anti-mouse F(ab′)2). d, Percentage of TCR+ and TCR− CAR+ cells. KI, knock-in; KO, knockout. e, Diagram of a B2M intron-targeted SEED HDRT encoding CD47. f–h T cells were edited with B2M intron-targeted RNP and HDRT (e) in the presence of M3814 then immunomagnetically purified with anti-B2M (n = 3 donors). f, Flow cytometry plots of B2M and CD47 expression. g, Percentage of B2M+ and B2M– CD47+ cells. h, Genomic DNA PCR targeting the SEED integration site at B2M. Amplicon for nonedited alleles, black triangle; amplicon for HDRT integration, blue triangle (n = 3 donors). No sel.; no selection; B2M+ dep.; B2M+ depleted. i, Fold enrichment of CD47+ SEED-edited cells relative to CD47− cells after coculture with NK cells (n = 3 T cell donors). Data are displayed as the mean ± s.e.m. Significance was assessed with a two-way analysis of variance (ANOVA) and Šidák’s multiple-comparisons test. LHA, left homology arm; RHA, right homology arms. APC, allophycocyanin; PE, phycoerythrin.
