Journal: Frontiers in Genome Editing

Article Title: CRISPR-mediated generation of a tumor-associated antigen-deficient Raji platform to investigate antigen loss in CAR-T cell therapy

doi: 10.3389/fgeed.2025.1649993

Figure Lengend Snippet: Genomic deletion of target antigens in Raji-Luc lines does not alter proliferation or viability. (A) Schematic representation of CRISPR-Cas9–mediated strategy to generate single, double, and triple knockout (KO) Raji-Luc cell lines. (B–D) Amplicon sequencing data showing genomic deletions at the CD19 (B) , CD20 (C) , and CD22 (D) loci in the respective KO lines, confirming targeted gene disruption. (E) Proliferation assay comparing all the Raji-Luc KO lines and control Raji-Luc-WT over 96 h. Data plotted as fold change normalized to Raji WT. (F) Viability analysis after 2 h incubation with AlamarBlue reveals no significant differences between Raji WT, Raji-Luc WT, and KO lines. Statistical analysis was performed using ordinary one-way ANOVA followed by the Dunnett’s multiple comparisons test. *, p < 0.05; ns = not significant.

Article Snippet: For CD19 and CD22 staining, cells were incubated with CD19-PE (Miltenyi Biotec) and CD22-APC (Miltenyi Biotec) antibodies for 30 min at 4 °C in the dark.

Techniques: CRISPR, Triple Knockout, Amplification, Sequencing, Disruption, Proliferation Assay, Control, Incubation

Journal: Frontiers in Genome Editing

Article Title: CRISPR-mediated generation of a tumor-associated antigen-deficient Raji platform to investigate antigen loss in CAR-T cell therapy

doi: 10.3389/fgeed.2025.1649993

Figure Lengend Snippet: Raji knockout lines generated via CRISPR-Cas9 exhibit complete loss of CD19, CD20, and CD22 transcript and protein expression. (A–C) Transcript level analysis via qRT-PCR revealed significant reduction in the mRNA levels of (A) CD19, (B) CD20 and (C) CD22. K562 was used as CD-marker negative control. Gene expression was normalized to Raji WT and GAPDH was used as an internal control. Data plotted as fold change (mean ± SD, n = 3). (D) Immunofluorescence staining for CD19 (yellow), CD20 (green), CD22 (red), and Hoechst (blue) in all Raji cell lines and K562. Loss of surface antigen staining is evident in all corresponding knockout lines. Images are representative of three independent experiments. Scale bar = 50 μm. (E–G) Flow cytometry analysis of surface CD19 (E) , CD20 (F) , and CD22 (G) expression in nonpermeabilized Raji-derived knockout cells and K562 control. Data represent mean ± SD of fluorescence intensity (MFI) from three independent experiments, confirming complete loss of surface expression of the respective target antigen in each knockout line. Statistical analysis was performed using ordinary one-way ANOVA followed by the Dunnett’s multiple comparisons test. ****, p < 0.0001; ***, p < 0.001; ns = not significant.

Article Snippet: For CD19 and CD22 staining, cells were incubated with CD19-PE (Miltenyi Biotec) and CD22-APC (Miltenyi Biotec) antibodies for 30 min at 4 °C in the dark.

Techniques: Knock-Out, Generated, CRISPR, Expressing, Quantitative RT-PCR, Marker, Negative Control, Gene Expression, Control, Immunofluorescence, Staining, Flow Cytometry, Derivative Assay, Fluorescence

Journal: Frontiers in Genome Editing

Article Title: CRISPR-mediated generation of a tumor-associated antigen-deficient Raji platform to investigate antigen loss in CAR-T cell therapy

doi: 10.3389/fgeed.2025.1649993

Figure Lengend Snippet: Raji KO lines model antigen loss and resist CAR-T cell–mediated cytotoxicity. (A) Schematic representation of CAR constructs targeting CD19, CD20, and CD22. (B) Flow cytometric analysis of isolated T cells confirmed the expression of T cell–specific markers and negligible expression of non–T cell markers. (C) Expression of T-cell activation markers on isolated T cells post 48 h of activation with CD3/CD28 beads. (D) Flow cytometry of GFP expression in T cells 48 h post-transduction. Histogram shown from one of three independent transductions using T cells from three different donors. (E–G) Viability of Raji KO lines assessed by bioluminescence following 16 h co-culture with CAR T cells expressing CAR19 (E) , CAR20 (F) or CAR22 (G) at indicated effector-to-target (E:T) ratios. Percent live cells was calculated based on luminescence signal relative to target-only control and normalized to untransduced controls. CAR-T cells selectively eliminated WT Raji cells expressing the corresponding target antigen, while respective single, double, and triple KO lines remained resistant. K562 cells served as antigen negative control. Data are presented as mean ± SD for three independent biological replicates. Statistical analysis was performed using multiple unpaired t-tests (one per row) with Holm–Šidák correction for multiple comparisons (C) or by comparing area under the curve for each cell line group in an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (E–G) . ***, p < 0.001; ****, p < 0.0001; ns = not significant.

Article Snippet: For CD19 and CD22 staining, cells were incubated with CD19-PE (Miltenyi Biotec) and CD22-APC (Miltenyi Biotec) antibodies for 30 min at 4 °C in the dark.

Techniques: Construct, Isolation, Expressing, Activation Assay, Flow Cytometry, Transduction, Co-Culture Assay, Control, Negative Control