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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: J Immunol. 2018 Jul 18;201(5):1586–1598. doi: 10.4049/jimmunol.1701616

FIGURE 1. CRISPR-mediated deletion of single or multiple genes in primary human T cells.

FIGURE 1

(A) CD4 and CD45 expression in purified human CD4+ T cells. CD4+ T cells were activated with anti-CD3/anti-CD28 beads and the next day transduced with entiviruses targeting CD45 gene (gCD45-g2), as described in methods. Cells were then expanded in IL-2 and stained at indicated time points with CD4 and CD45 antibodies. (B) Comparison of knockout efficiency using three different gRNAs targeting CD4 or CD45 gene loci in human CD4+ T cells with or without puromycin selection. Puromycin (0.4 μg/ml) was added at day 3 post-transduction to a portion of activated T cells infected with the lentiviruses. Cells were then stained for CD4 or CD45 expresssion at day 7 post-transduction (day 4 post-selection). Data represent three independent experiments with cells isolated from different donors. Error bars represent SEM. ***p<0.001. (C) Double gene deletions within the same T cell. CD4+ T cells were activated and transduced with either single (gCD4, gCD95 or gCD45 alone), double (gCD4 + gCD95 or gCD4 + gCD45) lentiviruses specifically targeting CD4, CD95, or CD45 gene. (D) Analysis of triple gene deletions. T cells were simultaneously transduced with gRNAs targeting three genes (gCD4 + gCD95 + gCD45). Expression of CD45 and CD95 was determined after gating on CD4+ or CD4− T cells (top panel) and expression of CD4 and CD95 is shown after gating on CD45+ or CD45− T cells (bottom panel). Expression of surface markers was analyzed 7 days post-transduction without selection. (E) CD45 expression in human CD8+ T cells transduced with CRISPR/Cas9 targeting of CD45 gene. Purified CD8+ T cells were activated with anti-CD3/anti-CD28 beads and transduced next day with lentiCRISPR v2, as in CD4+ T cells in figure 1. Cells were cultured for another 8 days post-transduction in IL-2, without selection and stained with CD8 and CD45 antibodies. Data represent three independent experiments with cells isolated from different donors. Error bars represent SEM. **p<0.01. (F) Inducible CRISPR deletion of CD45 gene in primary human T cells and Jurkat cell line. Jurkat and CD4+ T cells transduced with a lentivector encoding Cas9 under inducible Tetracycline promoter and gRNA targeting CD45 gene (gCD45-g1). The transduced Jurkat and CD4+ T cells were either treated with 1 μg/ml or 10 μg/ml doxycycline, respectively, one day post-transduction or left untreated. After 7 day expansion in culture, without selection, cells were stained with anti-CD45 antibody to determine deletion. Data represent two independent experiments.