Journal: Life Science Alliance
Article Title: STAG1 vulnerabilities for exploiting cohesin synthetic lethality in STAG2-deficient cancers
Figure Lengend Snippet: Extended validation of candidate genes displaying stronger dropout effects in STAG2 -null versus wild-type cells. (A) Cas9-GFP–expressing KBM-7 clonal cell line (B4) was lentivirally transduced with a validated sgRNA targeting STAG2 , and several subclones were isolated and characterized for STAG2 knockout by immunoblotting. (B) For each candidate gene, multiple sgRNAs scoring in the primary screen (2 or 3, as indicated) were tested in parallel competitive proliferation assays in STAG2 –wild-type (B4, blue lines) and STAG2 -mutant (c9, red lines) cell lines used in the screen and in an independent STAG2 -mutant clone (c11, orange lines). Cells were transduced with lentiviral vectors co-expressing mCherry and an sgRNA targeting the indicated genes, and the fraction of mCherry+ cells was monitored over time using flow cytometry. Plotted are averages of 2–3 independent sgRNAs analyzed for each gene; error bars represent standard deviations. Note that knockout of most candidates (e.g., BUB1, PAXIP1, PDCD5, PP2R1A, and SKA3) was deleterious in both STAG2–wild-type and STAG2-mutant cells, with some differences in depletion dynamics. In other instances, knockout effects turned out to be specific without being strongly detrimental in STAG2-mutant cells (e.g., CHMP7, FLI1, and SKI), and in some cases, they could not be confirmed in an independent STAG2-null clone (c11, e.g., DNAJC9 and FANCI), indicating that clonal artifacts remain an issue even when starting out from a single-cell–derived clone.
Article Snippet: 4 d postinfection, the transduction efficacy was determined using antibody staining for Thy1.1 (APC anti-mouse CD90.1, 202526; BioLegend) and flow cytometry analysis.
Techniques: Expressing, Transduction, Isolation, Knock-Out, Mutagenesis, Flow Cytometry, Derivative Assay