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. 2022 Apr 22;219(5):e20211530. doi: 10.1084/jem.20211530

Figure S1.

Figure S1.

Optimization of nonviral gene editing in primary T cells using plasmid-based donor templates. (A–F) Titration of linear dsDNA and nanoplasmid donor templates in CD8+ T cell cultures in RPMI/10% FBS medium. (A) Diagram of linear dsDNA knock-in construct TRAC-mNG. (B) Representative contour plots showing the frequency of CD8+ T cells expressing mNG. (C) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8+ T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of linear dsDNA donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range (n = 4). This experiment was performed twice. (D) Diagram of nanoplasmid knock-in construct TRAC-mNG. (E) Representative contour plots showing the frequency of CD8+ T cells expressing mNG. (F) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8+ T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range (n = 4). This experiment was performed twice. (G) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus in the presence of absence of PGA. Circles represent individual donors; bars represent median values with range (n = 3). This experiment was performed twice. (H) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template that either did or did not contain truncated Cas9 target sequences (tCTS) together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range (n = 3). This experiment has been performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 in RM one-way ANOVA with Geisser–Greenhouse correction (C and F) or paired t test (G and H).