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. 2019 Dec 23;8:e51163. doi: 10.7554/eLife.51163

Figure 6. A genome-wide CRISPR fitness screen identifies UBE2G1 as the primary genetic vulnerability of a UBE2R1/2 double knockout cell line.

(a) Schematic of CRISPR screen workflow to identify genes that cause synthetic lethality in a UBE2R1/2 double mutant background. (b) Scatter plot of differential RANKS scores of all genes in replicate UBE2R1/2 double knockout screens. Negative values indicate sgRNA depletion relative to the control population background and positive values indicate sgRNA enrichment. (c,d). Scatter plot comparisons of differential RANKS scores for UBE2R1/2 double knockout compared to UBE2R2 and UBE2R1 screens. For both plots, UBE2R1/2 scores are the average of the two screens in panel b. (e) Scatter plot comparison of UBE2R1 versus UBE2R2 screens. Scores for UBE2R2 screens are the average of two independent replicates, whereas scores for UBE2R1 are from a single screen.

Figure 6.

Figure 6—figure supplement 1. Knockout populations for control, UBE2R1 and UBE2R2 loci used in genome-wide CRISPR screens.

Figure 6—figure supplement 1.

NALM-6 populations treated with sgRNAs that target AAVS1, GFP, UBE2R1 and/or UBE2R2 as indicated. UBE2R1 and UBE2R2 protein were detected by immunoblot with anti-UBE2R1 or anti-UBE2R2 antibody at 1:1000 dilution. Note that since CRISPR-mediated knockout populations were not clonal, residual protein was detected in some instances.
Figure 6—figure supplement 2. Proliferation defect of UBE2R1/UBE2R2/UBE2G1 triple knockout NALM-6 cells.

Figure 6—figure supplement 2.

(a) Loss of UBE2R1 and UBE2R2 protein in clonal knockout NALM-6 cell lines for the UBE2R1 locus (top) or UBE2R2 locus (middle) or both loci (bottom). Clones used for analysis in panel b and c are indicated by red boxes. (b) Loss of UBE2G1 protein in AAVS1/GFP control and UBE2R1/UBE2R2 double knockout NALM-6 cell lines populations treated with indicated lentiviral constructs that target either control AZ-Green or UBE2G1. (c) Viability of colonies for the indicated combinations of UBE2R1, UBE2R2 and UBE2G1 knockouts. All cell lines and populations for validation experiments were generated in the NALM-6 doxycycline-inducible Cas9 clone #20 background.