LCL is more dependent on iron reductase STEAP3 than Burkitt B cells. (A) Distribution of Avana human genome-wide CRISPR screen sgRNA log₂ fold-change values at day 21 vs input in GM12878 (top) and P3HR-1 (bottom). Values for STEAP3 targeting sgRNAs are overlaid on gray gradients depicting all Avana sgRNA library values.³⁰ Average day 21 vs input values from 4 screen biological replicates are shown. (B) Growth curve analysis of GM12878 (left) and P3HR-1 (right) at the indicated time points after expression of control or independent STEAP3 sgRNAs and puromycin selection. Mean ± standard deviation values from n = 3 replicates are shown. (C) Indel-sequencing analysis of Cas9 activity at the STEAP3 locus. Shown are the percentage of indels in P3HR-1 or GM12878 cells expressing the indicated control or STEAP3 sgRNAs. (D) RNA-sequencing analysis of STEAP family messenger RNAs at the indicated days postprimary human B-cell EBV infection.³⁵ (E) Chromatin immunoprecipitation–sequencing tracks of the indicated Epstein-Barr nuclear antigens, NF-κB subunits, and H3K27 acetyl (H3K27ac) marks at the STEAP3 locus in LCLs. (F) Immunoblot analysis of WCLs from GM12878 (left) and P3HR-1 (right) 9 days after control or STEAP3 sgRNA expression. (G) Growth curve analysis of STEAP3 cDNA rescue effects on P3HR-1 (left) or GM12878 (right) with control or CYB561A3 sgRNAs. Shown are mean ± standard deviation values from n = 3 replicates. (H) Immunoblot analysis of WCL from GM12878 (left) and P3HR-1 (right) expressing the indicated control or CYB561A3 sgRNAs and cDNAs expressing green fluorescent protein (GFP) vs STEAP3. (I) Log₂ normalized RNA-sequencing STEAP3 (y-axis) vs CYB561A3 (x-axis) values from EBV⁺ (red) and EBV^– (blue) Burkitt lymphoma cell lines profiled by DepMap. Representative blots from n = 3 replicates are shown in panels F and H. *P < .05; **P < .01; ***P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant (unpaired Student t test).