Fig. 1: CRISPR screen in HBLAK cells: Workflow, data generation, and analysis.

a Representative images of 3D growth of HBLAK cells in ultra-low attachment plates. The scale bar represents 100 µm. b Plots illustrating the area of spheroids and CyQuant fluorescence readings over time. c Images showing that HBLAK cells do not grow but survive in the soft agar clonogenic assay while J82 cells are able to grow and form viable colonies. d The growth kinetics of HBLAK xenografts in FOXN−/− mice when injected at different cell numbers, showing a gradual decrease and eventually disappearance of the xenografts. M: million. e Workflow of the CRISPR screening in HBLAK cells. The cells were first transduced with spCas9 containing virus, which were re-infected with the DTKP Bassik library. The positively transfected cells, selected via antibiotic, were allowed to grow in 2D and 3D growth conditions. The surviving cells were sequenced to identify the sgRNAs that dropped out over time. f Workflow depicting how the FASTQ files of sequencing data were processed using the MAGeCK method to produce a phenotype score for each gene. g Scatter plots showing the diverse phenotype patterns in the 2D—day 14 and 3D—day 14 normalized against day 0 at respective growth setting.