Fig. 2: CRISPR-Cas9-engineered T-cells produced via a localized in vivo electroporation.

a A one-time treatment of focused in vivo lymph node electroporation lasting several minutes led to PD1-deficient T-cell production (day 7) in a minimally invasive surgery manner. b Flow cytometry analysis and (c) quantification data showing eGFP plasmid DNA transfection efficiencies with different in vivo delivery methods. d The qualification of eGFP transfection in different types of lymph node cells. Insert: The proportion of different lymphocytes within the eGFP-positive population. For (c, d), n = 4 mice in each group. Fluorescence microscopy images (e) and fluorescence qualification (f) of lymph node slice after PD1-CRISPR-Ele treatment versus untreated group. Scale bar, 100 µm. Scale bar, 10 µm in enlarged view. n = 5 independent samples. g Quantification of finely classified different T lymphocytes subsets. h Lymphocytes were isolated from the lymph nodes of mice 7 days after PD1-CRISPR-Ele treatment. Representative flow cytometry analysis of PD1 negative (PD1^-) cells population upon PD1-CRISPR-Ele treated group and untreated group. i Quantification of the proportion of PD1 positive (PD1⁺) cells in the CD3⁺ (T-cells) and CD3^- subsets (non-T-cells). n = 5 mice in each group. j, k Artificial ex vivo stimulation of lymph node cells (from healthy mice) to evaluate the PD1 knockdown efficiency. j Flow cytometry analysis and (k) qualification of PD1 expression in the CD3⁺ T-cells after a manual activation using the CD3/CD28 T-cell activator, n = 3 mice in each group. Flow cytometric analysis of PD1 expression on CD4⁺ T lymphocytes (l) and CD8⁺ T lymphocytes (m) extracted from the lymph nodes of different groups, n = 4 mice in each group. The data are presented as mean ± s.e.m. Statistical significance was calculated by two-sided Student’s t test for two group comparison, one-way ANOVA with Tukey’s test for multiple comparisons. Source data are provided as a Source Data file.