Supplementary Figure 1: CRISPRa in cancer cells induces stronger effector function of T cells by boosting the presentation of targeted antigens.

a, The expression of dCas9 protein in E0771-dCas9-VP64 and E0771-dCas9-VP64-MPH cell lines was confirmed by western blot using a monoclonal antibody against Cas9. Anti-GAPDH was used as an internal control. Representative data out of two independent experiments. b-c, OT-I CD8⁺ T effector cells after co-culture with E0771-OVA cancer cells. In each treatment group, n = 11 independent cell co-cultures, pooled from three independent experiments. b, Quantification of the geometric mean fluorescent intensity (MFI) of IFNγ in OT-I CD8⁺ T effector cells after co-culture with E0771-OVA cancer cells for 3h. Two sided Welch’s unpaired t-test: CRISPRa sgRNAs vs. vector in E:T = 1:1, p = 0.0713; CRISPRa sgRNAs vs. vector in E:T = 0.5:1, p = 0.0368. c, The percentage of cells undergoing apoptosis among all non-dead cancer cells after co-culture with OT-I CD8⁺ T effector cells for 24h. Two sided Welch’s unpaired t-test: CRISPRa sgRNAs vs. vector in E:T = 1:1, p = 0.001; CRISPRa sgRNAs vs. vector in E:T = 0.5:1, p = 0.002. d, Tukey boxplots (interquartile range (IQR) boxes with 1.5 × IQR whiskers) showing library representation of genome-scale mouse CRISPRa (SAM) library-transduced E0771 cells. n = 6 infection replicates from two independent experiments. Out of the 66,749 sgRNAs in the library, the six replicates cover 99.44% to 99.69% of the total sgRNAs. e, Tumor growth curves of E0771- Vector tumors that were treated with PBS (n = 5 independent mice), α-CD8 (n = 3), or c-MAEGI + α -CD8 (n = 3). Blue arrows, c-MAEGI treatment; brown arrows, α -CD8 treatment. Two-way ANOVA: PBS vs α-CD8, p = 0.9744; PBS vs. c-MAEGI + α-CD8, p = 0.6037; α-CD8 vs. c-MAEGI + α-CD8, p = 0.705. f, Representative peripheral blood flow cytometry analysis of CD8⁺ T cells after PBS or α-CD8 treatment, gated on CD45⁺CD3⁺ cells. Top row, PBS treatment; bottom row, α-CD8 treatment. Left column, DPI = 14; right column, DPI = 21. X-axis of scatterplots, CD4; Y-axis of scatterplots, CD8a. Percentages are noted in each quadrant. Over 50,000 PBMCs were analyzed for each sample. g, Bar plots detailing the % of CD8⁺ T cells among CD45⁺ PBMCs, 14 days after PBS or α-CD8 treatment. Data from PBS treated mice (n = 3), and α-CD8 treated mice (n = 4). Two-tailed unpaired t-test: PBS vs. α-CD8, p < 0.0001. h, Bar plots detailing the % of CD8⁺ T cells among CD45⁺ PBMCs, 21 days after PBS or α-CD8 treatment. Data from PBS treated mice (n = 4), and α-CD8 treated mice (n = 4). Two-tailed unpaired t-test: PBS vs. α-CD8, p < 0.0001. Error bars: Data points in this figure are presented as mean ± s.e.m. Asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001.