Cell Culture, shRNA Knockdown, and CRISPR/Cas9-Guided Gene Editing.

HCT116, Caco2, HT55, RKO, and LoVo cell lines were purchased from ATCC. HCT116 and RKO cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS); Caco2 and HT55 cells were grown in DMEM with 15% FBS; LoVo cells were cultured in Ham’s F12 with 10% FBS. The sequences of sgRNA, screen primers, KI donor sequences, and qRT-PCR primers are listed in SI Appendix, Table S1. Lentiviruses were packaged with psPAX2 and CMV-VSVG in 293T cells. Culture media was collected at 24 h and 48 h post transfection, passed through 0.45 µm filters, concentrated with lenti-X concentrator (Takara Bio), and resuspended in DMEM with 10% FBS. Cells were infected with lentiviruses and selected with puromycin (2 μg/mL) for 5 d before low-density seeding for picking single clones.