(A) Cartoon depicting the current model of the WNT/CTNNB1 pathway. In the absence of WNT ligands (left, ‘OFF’), free cytoplasmic CTNNB1 is captured by the destruction complex consisting of AXIN, APC, CSNK1A1, and GSK3, which leads to its phosphorylation, BTRC-mediated ubiquitination and subsequent proteasomal degradation, resulting in low levels of CTNNB1 in the cytoplasm and nucleus. Binding of the WNT protein (right, ‘ON’) to the FZD and LRP receptors inhibits the destruction complex through DVL. CTNNB1 accumulates in the cytoplasm and subsequently translocates to the nucleus, where it promotes the transcription of target genes, such as AXIN2, as a co-activator of TCF/LEF transcription factors. (B) Cartoon depicting exon 2 of the CTNNB1 locus, which contains the start codon, and the CTNNB1 protein before (top) and after (bottom) introduction of the SGFP2 by CRISPR/Cas9-mediated homology directed repair. (C) Schematic of the experimental workflow and timeline for generating HAP1SGFP2-CTNNB1 clones. Cas9, gRNA and repair templates are transfected as plasmids. The repair template contains the coding sequence of SGFP2 surrounded by 800 bp homology arms on either side and lacks the gRNA recognition site (see supplement 2 of this figure). A short puromycin selection step is included from 24 to 48 hr after transfection to enrich for transfected cells. Haploid, GFP-positive cells are sorted, and single cell clones are expanded for further analysis. (D–F) FACS plots illustrating control (D) and SGFP2-CTNNB1-tagged cells (E–F). (D) Cells transfected with Cas9 and gRNA in the absence of a repair template were used to set the gate for SGFP2-positive events. (E) A small population of cells expressing low levels of SGFP2 can be detected when cells are transfected with Cas9, gRNA, and repair template. (F) Treatment for 24 hr of cells similar to those depicted in (D) with 8 μM CHIR99021 does not change the amount of cells that are SGFP2 positive, but increases the SFP2 signal, most likely reflecting an increase in SGFP2-tagged beta catenin levels on a per cell basis and supporting the notion that the gated events indeed represent successfully tagged cells.