Extended Data Fig. 2: Non-viral genome targeting is consistent across T cell types and reproducible across target loci.

a, Efficient genome targeting was accomplished with a variety of T cell processing and handling conditions that are used with current manufacturing protocols for cell therapies. Non-viral genome targeting of a RAB11A–GFP fusion protein using a linear dsDNA HDR template was performed in bulk CD3⁺ T cells isolated from either whole blood draws or by leukapheresis. b, Targeting was similar either using bulk CD3⁺ T cells fresh after isolation or after cryopreservation (stored in liquid nitrogen and thawed before initial activation). c, CD4⁺ T cells isolated by FACS showed detectable GFP⁺ cells indicative of efficient editing, albeit at lower rates than targeting in CD4⁺ cells isolated by negative selection (potentially due to the added cellular stress of sorting). d, Using the same optimized non-viral genome targeting protocol (Methods), a variety of T cell types could be successfully edited, including peripheral blood mononuclear cells, without any selection (T cell culture conditions cause preferential growth of T cells from PBMCs). Sorted T cell subsets (CD8⁺, CD4⁺, and CD4⁺IL-2Rα⁺CD127^lo T_reg cells) could be successfully targeted with GFP integration. PBMCs were cultured for 2 days identically to primary T cells (Methods). Bulk CD3⁺ T cells were isolated by negative enrichment. The electroporations in d used only 2 µg of dsDNA HDR template, a concentration that was later found to be less efficient than the final 4 µg (contributing to the lower efficiencies seen compared to Fig. 1d). RAB11A–GFP template was used with on-target gRNA was used in a–d. e, Four days after electroporation of different GFP templates along with a corresponding RNP into primary CD3⁺ T cells from six healthy donors, GFP expression was observed across both templates and donors. f, High viability after electroporation was similarly seen across target loci. g, The fusion tagged proteins produced by integrating GFP into specific genes localized to the subcellular location of their target protein (Fig. 2b), and were also expressed under the endogenous gene regulation, allowing protein expression levels to be observed in living primary human T cells. Note how GFP tags of the highly expressed cytoskeletal proteins TUBA1B (beta tubulin) and ACTB (beta actin) showed consistently higher levels of expression compared to the other loci targeted across six donors. GFP mean fluorescent intensity (MFI) was calculated for the GFP⁺ cells in each condition/donor, and normalized as a percentage of the maximum GFP MFI observed. h, Gene fusions not only permitted the imaging and analysis of expression of endogenous proteins in live cells, but also could be used for biochemical targeting of specific proteins. For example, chromatin-immunoprecipitation followed by sequencing (ChIP–seq), and more recently CUT&RUN, have been widely used to map transcription factor-binding sites; however, these assays are often limited by the availability of effective and specific antibodies. As a proof-of-principle, we used anti-GFP antibodies to perform CUT&RUN analysis in primary T cells in which the endogenous gene encoding the crucial transcription factor BATF had been targeted to generate a GFP-fusion. Binding sites identified with anti-GFP CUT&RUN closely matched the sites identified with an anti-BATF antibody. Anti-BATF, anti-GFP and no-antibody heat maps of CUT&RUN data obtained from primary human T cell populations electroporated with GFP–BATF fusion HDR template (untagged cells were not electroporated). Aligned CUT&RUN binding profiles for each sample were centred on BATF CUT&RUN peaks in untagged cells and ordered by BATF peak intensity in untagged cells. Experiment in h was performed in two independent healthy donors.