Inactivation of COPS5 and UBE2M promotes lenalidomide resistance and alters cullin 4 neddylation and CRBN levels. (A) MM1.S-Cas9 cells expressing EGFP were infected with gRNAs targeting indicated genes and mixed at a 5:95 ratio with control gRNA-infected cells. The cells were then grown for 20 days in the presence of DMSO or 1 μM lenalidomide and the percentage of EGFP⁺ cells was quantified every 4 days using flow cytometry. Data points are average of 3 technical replicates and error bars represent a 95% confidence interval. (B) HEK293T single-cell heterozygous for frameshifting mutations in COPS5 and UBE2M were generated via CRISPR-Cas9 editing. Protein lysates were harvested and immunoblotted (IB) for the indicated proteins. Labels indicate neddylated and unneddylated forms of cullin 4A. Data are representative of 3 experimental replicates. (C) Quantification of CRBN levels blots from panel B across 3 experimental replicates. Values are normalized to control guide 1 (g1) and the respective β-actin loading controls. Error bars represent standard error of the mean.