Fig. 6: Experimental validation of the FMRE at the RMRP/CCDC107 locus.

a Genomic locus of the frequently mutated regulatory element (FMRE) at the non-coding RNA gene RMRP. The plot shows small mutations (SNVs, indels) in primary breast cancers (PCAWG) and metastases (HMF) (top), TOP2B-CTCF-RAD21 binding site (middle), corresponding ChIP-seq signals from representative experiments (black) and MACS2 peaks (pink) (bottom). b FMRE Mutations grouped by cancer types. c Locus overview with pan-cancer mutation frequency (top), FMRE and binding sites (middle), and adjacent genes (bottom). Triangles indicate sgRNAs used in genome editing. d Experimental validation. Two sgRNAs targeting the orthologous mouse FMRE were delivered by lentivirus using ultrasound-guided in utero injection into mouse embryos. In parallel, two sgRNAs targeting the human FMRE were delivered into MCF10A human mammary epithelial cell lines (not shown). e Genome editing of mouse FMRE causes earlier tumor onset in vivo. Kaplan-Meier plot compares tumor-free survival of mice having FMRE-targeted sgRNAs (red or purple lines) and mice having control sgRNAs (grey lines). Hazard ratio (HR) and P-value were derived from Cox proportional-hazards regression with Wald test. HR is shown with 95% confidence intervals. f Three-dimensional (3D) Matrigel growth assay of MCF10A cells compares control (Scr)-treated cells (left) and FMRE-mutated cells (right). One representative experiment for sgRNA sg6-1835p is imaged on day 28. g Differential expression analysis of FMRE-mutant MCF10A cells. Scatterplot shows differential gene expression from RNA-seq data of 2D (X-axis) and 3D assays (Y-axis) of edited MCF10A cells relative to Scr-treated controls. Genes were prioritized based on either joint up-regulation or down-regulation in 2D and 3D assays and significant genes are colored (merged FDR < 0.05 from DPM⁶⁴). Directional significance scores display up-regulated genes on the top or right and down-regulated genes on the bottom or left. Cancer genes from Cancer Gene Census are labelled. h Significantly enriched pathways visualized as an enrichment map. Significant pathways and processes are shown as nodes that that are connected into subnetworks if the pathways share many genes (family-wise error rate (FWER) < 0.05 from ActivePathways⁵³). Node color corresponds to transcriptomics evidence from the two types of growth assays. Source data are provided as Source Data files.