Primary human T cells with two modifications were enriched by gating on the cells that had at least one modification, and this effect was consistent across multiple combinations of genomic loci. viability and function. This permits individual or multiplexed modification of endogenous genes. First, we apply this strategy to correct a pathogenic mutation in cells from patients with monogenic autoimmune disease, demonstrating improved signaling function. Second, we replace the endogenous YM348 T cell receptor (and (Fig. 1a). Both cell viability and the efficiency of this approach were optimized by systematic exploration (Fig. 1b and Extended Data Fig. 1f-h) resulting in GFP expression in ~50% of both primary human CD4+ and CD8+ T cells. The method was reproducibly efficient with high cell viability (Fig. 1c, d, e). The system is YM348 also compatible with current manufacturing protocols for cell therapies. The method can be used with fresh or cryopreserved cells, bulk T cells or FACS-sorted sub-populations, and cells from whole blood or leukapheresis (Extended Data Fig. 2a-d). Open in a separate window Figure 1: Efficient non-viral genome targeting in primary human NMA T cells.a, HDR mediated integration of a GFP fusion tag to the housekeeping gene gene using non-viral targeting in primary human CD4+ and CD8+ T cells. d, Average efficiency with the RAB11A-GFP HDR template was 33.7% and 40.3% in CD4+ and CD8+ cells respectively. e, YM348 Viability (number of live cells relative to non-electroporated control) after non-viral genome targeting averaged 68.6%. Efficiency and viability were measured 4 days following electroporation. Mean of n=12 independent healthy donors displayed (d-e). See also Extended Data Fig 1. We next confirmed that the system could be applied broadly by targeting sequences in different locations throughout the genome. We efficiently engineered primary T cells by generating GFP fusions with different genes (Fig. 2a and Extended Data Fig. 2e-g). Live-cell imaging with confocal microscopy confirmed the specificity of gene targeting, revealing the distinct sub-cellular locations of each of the resulting GFP-fusion proteins11 (Fig. 2b). Appropriate chromatin binding of a transcription factor GFP-fusion protein was confirmed by performing genome-wide CUT & RUN12 analysis with an anti-GFP antibody (Fig. 2c and Extended Data Fig. 2h). Finally, we showed that gene targeting preserved the regulation of the modified endogenous gene. Consistent with correct cell-type specific expression, a CD4-GFP fusion was selectively expressed in the CD4+ population of T cells (Fig. 2d). Using HDR templates encoding multiple fluorescent proteins, we demonstrated that we could generate cells with bi-allelic gene targeting (Fig. 2e and Extended Data Fig. 3a-d) or multiplex modification of two (Fig. 2f and Extended Data Fig. 3e-h) or even three (Fig. 2g and Extended Data Fig. 3i) different genes13,14. These results show that multiple endogenous genes can be directly engineered without virus in T cells, and that gene and protein regulation are preserved. Open in a separate window Figure 2: Individual and multiplexed modification of endogenous T cell genes.a, Non-viral genome targeting with GFP-fusion constructs into multiple endogenous genes. b, Confocal microscopy of live human T cells electroporated with the indicated HDR templates confirmed fusion-protein localization. Scale = 5 m. c, GFP fused to the endogenous transcription factor BATF enabled genome-wide binding analysis (CUT&RUN) using anti-GFP or anti-BATF antibodies. d, RAB11A-fusions produced GFP positive CD4+ and CD8+ cells, whereas the CD4-fusions were selectively expressed in CD4+ cells. e, Bi-allelic non-viral genome targeting of two distinct fluorescent proteins into the same locus. f, Multiplexed non-viral genome targeting of HDR templates into two separate genomic loci. g, Simultaneous targeting of three distinct genomic loci. Cells positive for one (Q-II, Q-III) or two integrations (Q-IV), were highly enriched for a third HDR integration. One representative donor displayed from n=6 (a), n=4 (b, d-g), or n=2 (c) independent healthy donors. See also Extended Data Figs 2, ?,33. For therapeutic use of genetically modified T cells, integrated sequences should be introduced specifically without unintended disruption of other critical genome sites15. We performed targeted locus amplification (TLA) sequencing16 and found no evidence of off-target integrations above the assays limit of detection (~1% of alleles) (Extended Data Fig. 4a-b). We further assessed potential off-target integrations at the single cell level by quantifying GFP+ cells generated using a Cas9 RNP that cuts outside the homology site. Similar to what has been YM348 described.