Supplementary Figure 9: Quantification of AAV–Cpf1 and Cas9 RNP-based double-CAR knockin.

a, FACS quantification of percentages of CD19CAR and CD22CAR 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 Cpf1 CAR19;CAR22 double knockin: for CAR19⁺ cells, ***P < 0.001; for CAR22⁺ cells, ***P < 0.001; for CAR19⁺CAR22⁺ cells, ***P < 0.001. b, FACS quantification of percentages of CD19CAR and CD22CAR single- and double-positive cells shown in a column graph (infection replicates, vector n = 4; Cas9 CAR19;CAR22 double knockin, n = 6). Two-way ANOVA with Sidak’s multiple-comparisons test was used to assess significance (multiple-testing corrected). Vector versus Cas9 CAR19;CAR22 double knockin: for CAR19⁺ cells, ***P < 0.001; for CAR22⁺ cells, n.s. P = 0.8036; for CAR19⁺CAR22⁺ cells, n.s. P = 0.9084. c, Quantification of percentages of AAV–Cas9-mediated double knockin into TRAC and PDCD1 locus using TRAC-sg1 and PDCD1-sg2/sg3/sg4 (infection replicates, vector n = 4, TRAC sg1 + PDCD1 sg2 n = 6 and TRAC sg1 + PDCD1 sg3/sg4 n = 3). Two-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance (multiple-testing corrected). CAR19⁺ group: vector versus TRAC sg1 + PDCD1sg2/ sg3/sg4, ***P < 0.001. CAR22⁺ group: vector versus PDCD1-sg2, P = 0.678; vector versus PDCD1-sg3, P = 0.824; vector versus PDCD1-sg4, P = 0.885. CAR19⁺CAR22 + group: vector versus PDCD1-sg2, P = 0.439; vector versus PDCD1-sg3, P = 0.684; vector versus PDCD1-sg4, P = 0.493. Data are shown as mean ± s.e.m., plus individual data points on the graph.