Supplementary Figure 3: Demonstration of one-step simultaneous generation of HDR-based transgene knockin at the TRAC locus with PDCD1 knockout in human primary CD4⁺ T cells.

a, Schematic of a PDCD1^KO;dTomato-TRAC^KI AAV construct (TRAC-KIKO) and targeting strategy, where the dual crRNA array crTRAC;crPDCD1 generates double-strand breaks at both TRAC and PDCD1 loci, and an HDR donor (LHA-EFS-dTomato-PA-RHA) mediates dTomato integration into the TRAC locus. AAV6 was used for packaging. b, In-Out PCR for the targeted integration of dTomato at the TRAC locus. HDR and NHEJ products and percentage (quantified by gel-based method) are indicated by arrows pointing to bands of predicted sizes. c, The frequencies of dTomato HDR, TRAC NHEJ, and TRAC WT alleles in unsorted or CD3^–dTomato⁺ sorted T cells, measured by Nextera prep and deep sequencing (n = 3 infection replicates). Two-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance. WT group: vector versus unsorted, ***P < 0.001; vector versus sorted, ***P < 0.001; unsorted versus sorted, **P < 0.01. NHEJ group: vector versus unsorted, P = 0.2201; vector versus sorted, P = 0.2547; unsorted versus sorted, P = 0.9952. HDR group: vector versus unsorted, ***P < 0.001; vector versus sorted, ***P < 0.001; unsorted versus sorted, **P < 0.01. Data are shown as mean ± s.e.m., plus individual data points on the graph. d, Example reads of HDR mapping with dTomato TRAC knockin reference sequence. e, The PDCD1 cutting efficacy in unsorted or CD3^–dTomato⁺ sorted T cells, estimated by comparison to quantitative low-range DNA ladder as indicated below, and subsequently by using Nextera library preparation and Illumina sequencing. f, Indel analysis based on read-level quantification for PDCD1 knockout in unsorted or CD3^–dTomato⁺ sorted T cells (n = 3 infection replicates). One-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance. Vector versus unsorted, ***P < 0.001; vector versus sorted, ***P < 0.001; unsorted versus sorted, **P < 0.01. Data are shown as mean ± s.e.m., plus individual data points on the graph.