a, We wanted to confirm that we could generate cells with genome insertions in both alleles and quantify the frequency of bi-allelic modifications. Targeting the two alleles of the same gene with two distinct fluorophores would provide a way to quantify and enrich cells with bi-allelic gene modifications. The possible cellular phenotypes and genotypes when two fluorescent proteins are inserted into the same locus are displayed. Importantly, the number of cells that express both fluorescent proteins underestimates the percentage of cells with bi-allelic integrations because some cells will have inserted either GFP or mCherry on both alleles. We constructed a model to account for bi-allelic integrations of the same fluorescent protein (Supplementary Note 1). b, Diagram of bi-allelic integration model. The total percentage of cells with bi-allelic HDR integrations must be the sum of genotypes D, E and F. Although the proportion of cells with genotype E (dual fluor positives) is immediately apparent from the phenotypes, genotypes D and F are not. Our model allow for the de-convolution of the multiple genotypes in the single fluor positive phenotypes, and thus an estimation of the true percentage of cells bi-allelic for HDR. c, The observed level of bi-allelic integrations was higher in cells that acquired at least one integration than would be expected by chance. Individual points represent replicates where the combination of the genes encoding the fluorescent proteins was varied (either GFP plus mCherry, GFP plus BFP, or mCherry plus BFP) as was the amount of the HDR template (3–6 µg). d, Bi-allelic HDR analysis was applied across a variety of fluorophore permutations inserted into the RAB11A locus. e, Dual fluorescence bi-allelic integrations were seen across target loci. f, The data also suggest that cells with one integration were more likely to have also undergone a second targeted bi-allelic integration, and this effect was observed across three genomic loci. While the total percentage of cells with an insertion varied with the efficiency of each target site, the fold enrichment in the observed percentage of homozygous cells over that predicted by random chance was largely consistent across loci. g, Co-delivery of three fluorescent tags targeting the RAB11A locus resulted in only a few cells that expressed all three fluorophores, consistent with a low rate of off-target integrations. As a maximum of two targeted insertions are possible (at the two alleles of the locus; assuming a diploid genome), no cells positive for all three loci should be observed (triple positives). Indeed, while large numbers of single fluorophore integrations were observed (single positives), as well as cells positive for the various permutations of two fluorophores (double positives), there was an approximately 30-fold reduction in the number of triple positive cells compared to double positives. All flow cytometric analysis of fluorescent protein expression shown here was performed 4 days after electroporation. h, Multiplex editing of combinatorial sets of genomic sites would support expanded research and therapeutic applications. We tested whether multiple HDR templates could be co-delivered along with multiple RNPs to generate primary cells in which more than one locus was modified. 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. HDR template permutations from a set of six dsDNA HDR templates (targeting RAB11A, CD4 and CLTA; each site with GFP or RFP) were electroporated into CD3+ T cells isolated from healthy human donors. Four days after electroporation of the two indicated HDR templates along with their two respective on-target RNPs, the percentage of cells positive for each template was analysed by gating on cells either positive or negative for the other template. Not only was two-template multiplexing possible across a variety of template combinations, but gating on cells positive for one template (template 1+ cells, blue) yielded an enriched population of cells more likely to be positive for the second template compared to cells negative for the first (template 1− cells, black). 2 µg of each template, along with 30 pmol of each associated RNP, were electroporated for dual multiplexing experiments. i, We also achieved triple gene targeting and could enrich for cells that had a third modification by gating on the cells with two targeted insertions, an effect again consistent across target genomic loci. 1.5 µg of each template (4.5 µg total) were electroporated together with 20 pmol of each corresponding RNP (60 pmol total). Graphs display mean and s.d. in n = 4 (f–i) independent healthy donors. Other experiments (c–e) were performed in two independent healthy donors.