CRISPR-Mediated Generation of Evi-Deficient HCT116 Cells

HCT116^Evi_KO cells were generated using CRISPR/Cas9 following a protocol described in reference 44 (link). In short, the HCT116 cells were transfected with px459 vector (a gift from Feng Zhang, Addgene plasmid # 48139; http://n2t.net/addgene:48139; RRID: Addgene_48139) expressing Cas9 and sgRNA targeting human Evi/Wls (5′-GTAAGCCAGGGAAACGTCCA-3′). After 48 h, the cells were selected with 2 μg/ml of puromycin (Invitrogen) for 72 h. Because Evi silencing in HCT116 cells resulted in growth inhibition (16 (link)), either recombinant Wnt3a (50 ng/ml; R&D Systems) and/or medium from parental cell lines was added to the growth medium. Single-cell clones were selected and were used for further analysis. The generation of HCT116^Evi_KO cells will be described in detail in Voloshanenko et al (manuscript in preparation).