Extended Data Fig. 1: Development of non-viral genome targeting in primary human T cells.

a, Except where noted otherwise, ‘viability’ refers to the number of live cells in an experimental condition (expressed as a percentage) relative to an equivalent population that went through all protocol steps except for the actual electroporation (no electroporation control). ‘Efficiency’ refers to the percentage of live cells in a culture expressing the ‘knocked-in’ exogenous sequence (such as GFP). Finally, the total number of cells positive for the desired modification was calculated by multiplying the efficiency by the absolute cell count. Methodological changes that maximized efficiency were not always optimal for the total number of positive cells, and vice-versa. b, dsDNA, both circular (plasmid) and linear, when electroporated into primary human T cells, caused marked loss in viability with increasing amounts of template. Co-delivery of an RNP caused less reduction in viability post electroporation. Notably, no loss in viability was seen with ssODNs. c, RNPs must be delivered concurrently with DNA to see increased viability. T cells from two donors were each electroporated twice with an 8 h rest in between electroporations. Although two closely interspersed electroporations caused a high degree of cell death, delivery of the RNP and linear dsDNA template could be delivered separately. Initial RNP electroporation did not protect from the loss of viability if dsDNA was delivered alone in the second round of electroporation. d, We determined whether the order of adding reagents influenced targeting efficiency and viability. In wells in which the RNP and the DNA HDR template were mixed together before adding the cells (1. RNP + HDRT; 2. + Cells), there was a marked increase in targeting efficiency. e, Note, with the high concentration of dsDNA used in these experiments, viability was higher if the RNP and cells were mixed first and the DNA template was added immediately before electroporation (1. RNP + Cells; 2. + HDRT). Taken together, these data suggest that pre-incubation of the RNP and HDR template, even for a short period, increased the amount of DNA HDR template delivered into the cell, which increased efficiency but decreased viability. However, viability after RNP and dsDNA HDR template pre-incubation was still higher than was observed with dsDNA HDR template electroporation by itself (b). dsDNA HDR temple (5 µg) was used in c–e. f, Primary human T cells were cultured for 2 days using varying combinations of anti-CD3/CD28 TCR stimulation and cytokines before electroporation of RAB11A targeting RNP and HDR template, followed by varying culture conditions after electroporation. g, Among the RNP and HDR template concentrations tested here, optimal GFP insertion into RAB11A was achieved at intermediate concentrations of the RNP and dsDNA HDRT. h, Arrayed testing of electroporation pulse conditions showed that, in general, conditions yielding higher HDR efficiency decreased viability. EH115 was selected to optimize efficiency, while still maintaining sufficient viability. i, Diagrammatic timeline of non-viral genome targeting. Approximately one week is required to design, order from commercial suppliers, and assemble any novel combination of genomic-editing reagents (gRNA and the HDR template). Two days before electroporation, primary human T cells isolated from blood or other sources (Extended Data Fig. 2) are stimulated. dsDNA HDR templates can be made easily by PCR followed by a SPRI purification to achieve a highly concentrated and pure product suitable for electroporation. On the day of electroporation, the gRNA (complexed with Cas9 to form an RNP), the HDR template, and collected stimulated T cells are mixed and electroporated, a process taking approximately 1.5 h. After electroporation, engineered T cells can be readily expanded for an additional 1–2 weeks. Viability was measured 2 days after electroporation and GFP expression was measured at day 4. Graphs display mean (b, c, g, h) and/or individual donor values (b–h) in n = 2 independent healthy donors (b–h). For d, e and h, one representative donor is shown.