Supplementary Figure 7: Modular combinations of AAV–Cpf1-mediated efficient dual-CAR knockin and knockout in human primary CD4⁺ T cells.

a, Schematics of construct design for TRAC^KO;CD19BBz-PDCD1^KI (CD19BBz-KIKO) for AAV–Cpf1mediated CD19BBz transgene knockin into PDCD1 locus with TRAC knockout. b, Representative flow cytometry plots showing PDCD1 CD19BBz knockin 8 d after AAV transduction (AAV6 packaging). c, Quantification of CD19BBz knockin frequency at PDCD1 target site shown in a column graph (uninfected n = 2 cell culture replicates; AAV vector n = 3 infection replicates; TRAC^KO;CD19BBz-PDCD1^KI n = 3 infection replicates). Unpaired two-sided t test was used to assess significance. Vector versus CD19BBz-KIKO, ***P < 0.001. Data are shown as mean ± s.e.m., plus individual data points on the bar graph. d, Schematics of construct design for using both PDCD1^KO;CD22BBz-TRAC^KI and TRAC^KO;CD19BBz-PDCD1^KI for dual targeting. e, Representative flow cytometry plots of double knockin 5 d after transduction for both AAV constructs (AAV6 packaging). f, Quantification of percentages of CD19BBz and CD22BBz single- and double-positive cells shown in a dot-plot graph (infection replicates, n = 3). Two-way ANOVA with Sidak’s multiple-comparisons test was used to assess significance (multiple-testing corrected). Vector versus dual targeting, ***P < 0.001. Data are shown as mean ± s.e.m., plus individual data points on the graph. g, Analysis of TCR knockout at integrated cells by FACS. Quantification of TCR^– cells in different quadrants shown in a dot-plot graph (infection replicates, n = 3). Unpaired two-sided t-test was used to assess significance. TCR^– population, vector versus dual targeting, **P < 0.01, ***P < 0.001. Data are shown as mean ± s.e.m.