Utilizing a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation activity in colorectal cancer
Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators that regulate these key cellular programs in CRC, we developed an endogenous reporter system by genome-editing human CRC cell lines with knock-in fluorescent reporters at the SOX9 and KRT20 locus to report aberrant stem cell-like activity and differentiation respectively in pooled CRISPR screening formats. We then constructed a dual reporter system that simultaneously monitors aberrant stem cell-like and differentiation activity in the same CRC cell line, improving our signal to noise discrimination. Using a focused-library CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs, we identified factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominated SMARCB1 of the SWI/SNF BAF complex as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency in CRC and required for human CRC cell line and patient-derived organoid growth in vivo. These studies highlight the utility of a biologically designed endogenous reporter system to uncover novel therapeutic targets for drug development. Overall design: To monitor aberrant stem cell-like (SOX9) and differentiation (KRT20) programs within the same cell in colorectal cancer, we established a dual endogenous reporter cell lines by knocking in fluorescent reporters after marker genes (HT29 SOX9-mKate2/KRT20-GFP) for functional genetic screens. We then compared differential guide abundance using sgRNA read counts in each sorted cell fraction using flow cytometry (mKate2high/GFPhigh, mKate2high/GFPlow, mKate2low/GFPhigh, mKate2low/GFPlow).