Extended Data Fig. 1: Schematic overview of gastric organoid cultures, assays and sequencing time points.

Organoids were established from three donors (abbreviated D) as wild-type (WT) cultures. For each donor (D1-D3), three independent CRISPR/Cas9 edited TP53^−/− or TP53^−/−, APC^−/− cultures (abbreviated C) were established (indicated by sg 1-3) and referred to as C1-C3 (Methods). The WT and genome edited cultures were evolved under defined conditions for over two years. Sequencing was performed across the experimental time course at defined intervals: Early (~0–200 days), Mid (~200–400 days), Late (~400–900 days). Each original culture was thawed (indicated by dashed lines) at an Early/Mid (190–290 days) and Late (540–730 days) time point for additional replication and comparisons. The thawed samples were treated with normocin to eliminate mycoplasma (Methods). All cultures were subject to shallow WGS (sWGS). A subset of cultures underwent deeper WGS and/or single cell RNA (scRNA)-sequencing at select time points. In addition to these non-barcoded cultures, representative TP53^−/− HGO cultures from each donor were selected for prospective lineage tracing via transduction of a lentiviral expressed cellular barcode (ECB), as indicated by the multi-coloured circle in the legend. These ECB cultures were similarly subject to sWGS and scRNA-seq. Broad time intervals are indicated as in the legend, while days in culture are provided for individual cultures. Note that scRNA for D1C3 “Mid trajectory” was sampled at day 413.