Extended Data Fig. 1: High quality and reproducibility of 2D and 3D genome-wide CRISPR screens and hits with differential effects in the two conditions.

a, H23 cells expressing mCherry were seeded at different densities in ultra-low attachment plates in the presence of 0.75% methylcellulose. Sytox Green was added at 100 nM concentration. Average mCherry signal and Sytox Green signal measured across single cells were used to estimate the total numbers of live cells and dead cells at each seeding density. Cell growth and death rates were then monitored simultaneously on a live-cell microscope for 60 h. We aimed for a cell death rate of about 30% during the initial growth phase of spheroids, and 10⁵ cells per well (1.9 cm²) was the chosen cell seeding density for our genome-wide screens in 3D spheroids. b, Two-dimensional growth phenotypes of 20,463 genes were highly reproducible between experimental replicates (top). Sequencing counts of 208,687 sgRNAs in a T0 sample and a day 21 sample from the 2D genome-wide screens (bottom) show that most negative-control sgRNAs (red dots) are not enriched or disenriched between T0 and day 21 (black dots). This indicates the complexity of the genome-wide library was maintained throughout the 2D screen. In the top plot, the data are fit by a linear regression line (blue dotted line). The grey line marks a 1:1 diagonal. c, The quality and reproducibility of the 3D screens were comparable to those of the 2D screens, suggesting that the scalable 3D spheroid culture system is on a par with traditional 2D culture methods for its performance in genome-scale CRISPR screens. n = 20,463 genes (top); n = 208,687 sgRNAs (bottom). In the top plot, the data are fit by a linear regression line (blue dotted line). The grey line marks a 1:1 diagonal. d, Cumulative distribution of sequencing reads for sgRNAs in the genome-wide CRISPR library. Read counts were normalized by total reads for each sample and the cumulative sums of sgRNAs were plotted as relative percentages of the number of expected sgRNAs. e, Cumulative sums of TSG counts (left) or oncogene counts (right) are plotted against genes sorted by their 2D, 3D or 3D/2D phenotypes (T-score) from the genome-wide screens in H23 cells. TSGs are expected to have positive growth phenotypes when deleted. Therefore, genes are sorted in descending order from the most positive to the most negative phenotypes in the left plot. Oncogenes are expected to have negative or toxic growth phenotypes and genes are sorted in ascending order in the right plot. Black dotted line, randomly sorted genes. The first 4,000 genes are displayed. f, Summary of hits with differential 3D/2D phenotypes. Top positive (red-filled circles) and negative (blue-filled circles) hits from the differential 3D/2D phenotypes reveal many cancer-relevant genes associated with transcriptional regulation, cell motility, cell adhesion and energy metabolism. Cancer-signalling pathways such as Ras–MAPK, TGFβ, MET, Rho, β-catenin and Hippo signalling are highly represented. Sizes of circles are proportional to 3D/2D phenotype scores. g, The 10 most significant pan-lung cancer genes³¹ and 50 top core essential genes are marked. Genes sorted by absolute phenotype (T-score) in 2D, 3D and 3D/2D (see Methods).