Extended Data Fig. 7: Genome-scale CRISPR screening identifies BATF as an important REGNASE-1 functional target in tumour immunity.

a, Scatter plot of the enrichment of each gene versus its adjusted P value in genome-scale CRISPR screening. Gene enrichment was calculated by averaging the enrichment of the corresponding sgRNAs (n = 4 for each gene) in tumour-infiltrating OT-I cells relative to input (log₂(TIL/input ratio)), with the most extensively enriched (red) and selectively depleted (blue) genes (adjusted P < 0.05), as well as dummy genes (green, generated by random combinations of 4 out of 1,000 non-targeting control sgRNAs per dummy gene). b, Venn diagram showing the overlap of genes between top-depleted genes in genome-scale CRISPR screening (by less than −3.5 log₂(TIL/input ratio); adjusted P < 0.05) and top-upregulated genes in TIL Regnase-1-sgRNA- versus control-sgRNA-transduced OT-I cells as identified by RNA-seq (by greater than 1.5 fold change (log₂-transformed ratio); adjusted P < 0.05). c, Tn5 insert sites from ATAC-seq analysis were aligned to motifs for transcription factors from the TRANSFAC database, and the binding profiles of BATF are shown. d, Enrichment of BATF-binding motifs in the genomic regions with upregulated accessibility in REGNASE-1-null cells. First, we analysed common regions in our REGNASE-1-null ATAC-seq data and published BATF ChIP-seq peaks (GSE54191²⁶). Next, we scanned these common regions with TRANSFAC motifs for BATF, and numbers of motif matches and associated Fisher’s exact test P values and log₂(odds ratios) are shown (a positive log₂(odds ratio) value indicates that a motif is more likely to occur in REGNASE-1-null cells than in wild-type samples; ‘E−x’ denotes ‘ × 10^−x’). e, Luciferase activity of HEK293T cells measured at 48 h after transfection with Il2 mRNA 3′ UTR (top) or Il4 mRNA 3′ UTR (bottom) luciferase reporter plasmid, together with control (mock), wild-type REGNASE-1- or REGNASE-1(D141N)-expressing plasmid (n = 3 samples per group). f, OT-I cells transduced with control sgRNA (mCherry⁺; spike) were mixed at a 1:1 ratio with cells transduced with control sgRNA (ametrine⁺), Regnase-1 sgRNA (ametrine⁺), Batf sgRNA (GFP⁺) or Batf and Regnase-1 sgRNAs (GFP⁺ and ametrine⁺), and transferred into tumour-bearing hosts individually (n = 4 mice per group). Mice were analysed at 5 days after adoptive transfer for quantification of relative MFI of BATF normalized to spike in the tumour-infiltrating OT-I cells (f). g, Immunoblot analysis of REGNASE-1 and BATF expression in in vitro cultured OT-I cells 3 days after transduction with control sgRNA or Batf and Regnase-1 sgRNAs. HSP90 is used as a loading control. h–k, The same transfer system as in f was used. Five days after adoptive transfer, mice were analysed for the quantification of relative OT-I cell percentage in CD8α⁺ cells normalized to spike in the spleen (h, left, n = 4) and TILs (h, right, n = 4). Tumour-infiltrating OT-I cells were analysed at day 5 (n = 4 mice per group) for the quantification of the relative frequency of active caspase-3⁺ cells normalized to spike (i), and the quantification of the relative frequency of TCF-1⁺ cells normalized to spike (j), or at day 7 (n = 6 mice per group) for quantification of the relative frequency of IFNγ⁺ cells normalized to spike (k). l, Four million pmel-1 cells transduced with Regnase-1 sgRNA (ametrine⁺) (n = 10 recipients) or Batf and Regnase-1 sgRNAs (GFP⁺ and ametrine⁺) (n = 10 recipients) were transferred into mice at day 12 after B16 F10 melanoma engraftment, followed by analysis of tumour size. Mean ± s.d. (e). Mean ± s.e.m. (f, h–k). *P < 0.05, **P < 0.01, ***P < 0.001. Two-tailed unpaired Student’s paired t-test followed by Bonferroni correction (a), two-sided Fisher’s exact test (d), one-way ANOVA (e, f, h–k) or two-way ANOVA (l). Data are representative of two (e) or three (g) independent experiments, or pooled from two (f, h–l) independent experiments.

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