Supplementary Figure 8: AAV–Cas9 RNP–mediated single-CAR knockin and knockout in human primary CD4+ T cells with different guide RNAs.
From: One-step generation of modular CAR-T cells with AAV–Cpf1
a, Representative flow cytometry plots of TRAC CD22BBz knockin after 5 d using TRAC-sg1 and PDCD1-sg2 (AAV6 packaging). b, Quantification of CD22BBz knockin frequency at TRAC target site using TRAC-sg1 and PDCD1-sg2/sg3/sg4 5 d after AAV transduction (infection replicates, AAV vector n = 4; TRAC sg1 + PDCD1 sg2 n = 6; TRAC sg1 + PDCD1 sg3/sg4 n = 3). AAV vector sample was infected only with AAV6 carrying the same construct, and did not receive electroporation of Cas9 RNP thereafter. One-way ANOVA with Tukey’s multiple-comparisons test was used to assess significance. Vector versus CD22BBz KIKO, ***P < 0.001. c, Representative flow cytometry plots showing PDCD1 CD19BBz knockin after 5 d with TRAC-sg1 and PDCD1-sg2 (AAV6 packaging). d, Quantification of CD19BBz knockin frequency at TRAC target site using TRAC-sg1 and PDCD1-sg2/sg3/sg4 5 d after AAV transduction (infection replicates, AAV vector n = 4; TRAC sg1 + PDCD1 sg2 n = 6; TRAC sg1 + PDCD1 sg3/sg4 n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used to assess significance. Vector versus TRAC sg1+ PDCD1 sg2, ***P < 0.001. Vector versus TRAC sg1 + PDCD1 sg3, n.s. P = 0.1331. Vector versus TRAC sg1+ PDCD1 sg4, *P < 0.05. Data are shown as mean ± s.e.m., plus individual data points on the bar graph.
