Journal: Nature Communications

Article Title: Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor

doi: 10.1038/s41467-026-72846-2

Figure Lengend Snippet: A Polyfunctional editing strategy for the epi-silencing of B2M and TET2 and insertion of the CD19–28ζ CAR:ΔLNGFR construct into TRAC . B Left: percentages of the indicated T cell populations at day 14 after poly-editing (mean ± SD of 7 blood donors). TCR + : TCR-positive cells (green bar). TCR - : TCR-negative cells (light blue bar). TCR - /ΔLNGFR + : cells negative for endogenous TCRs and positive for ΔLNGFR (lilac bar). B2M - : B2M-negative cells (gray bar). B2M + : B2M-positive cells (white bar). Right: representative flow cytometry dot plots of poly-edited T cells showing expression of the TCRs and ΔLNGFR (left plot) and, within the TCR - /ΔLNGFR + cells, of B2M (right plot). C Fold-change in TET2 (light blue bars) and B2M (gray bars) expression in poly-edited vs . mock-treated cells (mean ± SD of 7 blood donors). D Schematic of the in vivo experiment. NSG mice were injected with GLuc.NALM-6 cells and, 7 days later, with the indicated T cell populations. Peripheral blood was collected at the indicated time points to measure tumor growth, phenotype human T cells, and quantify cytokine release. Mock: mock-transfected T cells. CAR-T Δ LNGFR : T cells expressing CD19–28ζ CAR:ΔLNGFR from TRAC . Poly-edited cells: T cells expressing CD19–28ζ CAR:ΔLNGFR from TRAC with epi-silencing of B2M and TET2 . E Tumor growth (left; mean ± SEM) and survival (right) curves over 20 days post-transplantation of the indicated T cell populations. n = 6 mice for Mock; n = 12 mice for each other group. ** p = 0.0075, *** p = 0.0008 by Mantel-Cox (log-rank) test. F Circos plots from CAST-Seq analyses of triple KO (left) and poly-edited (right) T cells ( n = 2 experimental replicates). Triple-KO cells were transfected with mRNA encoding Cas9 and the TRAC gRNA, together with gRNA B#6 and gRNA TE#19 . For poly-editing, the TRAC gRNA and the selected guide combinations for B2M and TET2 were used. Aberrations at the TRAC locus are in violet; translocations between TRAC and either B2M or TET2 are in blue; translocations between TRAC and OT sites of all gRNAs are in gray. G Number of unique CAST-Seq reads corresponding to either aberrations (violet bars) or translocations (gray bars) at the TRAC locus in the indicated treatments. Graphs were generated using GraphPad Prism (GraphPad Software). Source data are provided as a file.

Article Snippet: LNGFR; 130-113-422, Miltenyi), CD3 (345763, BD Biosciences), HLA-ABC (565332, BD Biosciences), HLA-E (130-117-402, Miltenyi), and CD19 CAR FMC63 Idiotype (130-127-342, Miltenyi).

Techniques: Construct, Flow Cytometry, Expressing, In Vivo, Injection, Transfection, Transplantation Assay, Generated, Software

Journal: Nature Communications

Article Title: Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor

doi: 10.1038/s41467-026-72846-2

Figure Lengend Snippet: A Scatter plot comparing whole-transcriptome analyses of T cells treated with the tripartite ETR and either full-length or truncated gRNAs targeting CD3D (left) or TGFBR2 (right) ( n = 2 experimental replicates). Data are expressed as log 2 TPM of mapped reads. B Polyfunctional editing strategy used to epi-silence CD3D and TGFBR2 and insert the scHLA-E:CD19–28ζ CAR cassette into exon 1 of B2M . C Left: percentages of the indicated T cell populations as measured by flow cytometry 14 days after polyfunctional editing (mean ± SD of 7 blood donors). HLA-ABC + : HLA-ABC-positive cells (light yellow bars); HLA-ABC - : HLA-ABC-negative cells (blue bars); CD19-CAR - : cells negative for the expression of the CD19–28ζ CAR (gray bars); CD19-CAR + : cells positive for the expression of the CD19–28ζ CAR (lilac bars); CD3 + : CD3-positive cells; CD3 - : CD3-negative cells; HLA-E - : HLA-E-negative cells; HLA-E + : HLA-E-positive cells. Right: representative flow cytometry dot plots of poly-edited T cells showing the expression levels of the HLA-ABC and CD3 (left plot) and, within the double-negative cells, of HLA-E and CD19-CAR (right plot). D Fold-change in the expression levels of CD3D (green bars) and TGFBR2 (orange bars) in poly-edited cells relative to mock-treated cells, 14 days post-editing (mean ± SD of 7 blood donors). Graphs were generated using GraphPad Prism (GraphPad Software). Source data are provided as a file.

Article Snippet: LNGFR; 130-113-422, Miltenyi), CD3 (345763, BD Biosciences), HLA-ABC (565332, BD Biosciences), HLA-E (130-117-402, Miltenyi), and CD19 CAR FMC63 Idiotype (130-127-342, Miltenyi).

Techniques: Flow Cytometry, Expressing, Generated, Software