Fig. 4: FUS-CRISPR-mediated telomere disruption can inhibit tumor cell growth and its resistance to CAR-T cell killing.

a Nuclear distribution of tagBFP-TRF2 and HaloTag-53BP1 in FUS-CRISPR-engineered HEK 293 T cells with telomere-targeting gRNA or non-targeting (NT) gRNA. HS, with 30 min HS; CT, without HS. Right, enlarged image merging TRF2 and 53BP1 signals. Scale bar = 10 μm. b Normalized cell number of FUS-CRISPR-engineered Nalm6 cells with telomere-targeting gRNA or NT gRNA two (D2) or four (D4) days after HS. Cell number was normalized to Day 0. N = 4 biological replicates. c Heat-map of differential gene expression in Nalm6 cells engineered with telomere-targeting or NT FUS-CRISPR at 24, 48, or 96 h after HS. d The top three enriched GO terms in the HS group compared to the CT group in the telomere-targeting FUS-CRISPR cells in (c). e Volcano plot showing the downregulated (blue) and upregulated (red) genes between HS and CT groups in the telomere-targeting FUS-CRISPR cells in (c). f Schematic illustration of CAR-T cell attack on tumor cells. g Survival (%) of FUS-CRISPR-engineered Nalm6 tumor cells 72 h after culture with (w/T) or without (w/o T) αCD19CAR-T cells in the luciferase-based cytotoxicity assay. The survival (%) was normalized to CT, w/o T group. P = 7.62 × 10⁻⁸ for w/o T, 5.32 × 10⁻⁵ for w/ T. h Cytotoxicity (%) of CAR-T cells in the co-culture groups (w/ T) in (g). The cytotoxicity (%) was quantified as 100% – Tumor survival (%). In (g and h), n = 3 technical replicates. Data are representative of two independent experiments. In (b, c, g, and h) HS: with 10 min HS; CT, without HS. Bar heights represent means; error bars represent s.e.m. Two-way ANOVA followed by Sidak’s multiple comparisons test. Source data are provided as a Source Data file. Created in BioRender. Liu, L. (2024) https://BioRender.com/c41g432.