Fig. 1
CRISPR-Cas9 knockout screen identifies Smarca5 as genetic dependency for SMB21 cells. (a) Illustration of CRISPR-Cas9 negative selection screening timeline for SMB21 cells. Illustration created with Biorender.com. Volcano plot showing the log2 fold change per gene and their associated negative score as determined by MAGeCK-RRA. Selected dependencies are denoted as colored data points. (b) Box plots demonstrating SMARCA5 expression in MB, CNS tumors and normal tissue, as determined from Griesinger (left panel) and Cavalli (right panel) publicly available datasets. Whiskers represent minimum and maximum points across all data points per group. The confidence intervals of the means for both plots are as follows: CIMB= 11.82–12.24, CINormal tissue= 10.82–11.45, CIOther tumors= 11.48–11.69, CISHH= 9.787–9.882, CIWNT= 9.516–9.620, CIGroup3= 9.307–9.407 and CIGroup4= 9.396–9.460. One-way ANOVA, Tukey´s multiple comparisons test. (c) Western blotting depicting SMARCA5 protein expression in sgSmo, sgSmarca5, and control SMB21 cells (left panel). Corresponding bar graph illustrates quantification of SMARCA5 (right panel). (d) Analysis of cell population doublings of SMB21 cells transduced with the indicated sgRNAs, as compared to SMB21 parental cells over 7 days. Two-way ANOVA, Tukey´s multiple comparisons test (n = 4). (e) Western blotting depicting GLI1 protein levels in sgSmo, sgSmarca5, and control SMB21 cells (left panel). Quantification of GLI1 protein levels in cells shown is indicated in the right panel. One-way ANOVA, Tukey´s multiple comparison test (n = 4). All graphs display mean ± SD. *p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
