Protein-domain based CRISPR-Cas9 screen identifies SPOP as a novel fetal globin repressor. (A) Screening strategy. Cas9-expressing HUDEP-2 cells were transduced with a BTB domain and histone modification reader domain-targeting sgRNA library (6 sgRNAs per domain). Edited HUDEP-2 cells were then induced to differentiate for 7 days. Differentiated cells were stained by allophycocyanin (APC)-conjugated anti-HbF and sorted into HbF-high and HbF-low cells by FACS. Enriched sgRNAs were identified by deep sequencing. (B) HbF FACS gating strategy for HbF-high and HbF-low cell populations. (C) Scatter plot of HbF-high (y-axis) and HbF-low (x-axis) populations as log₂ transformed normalized read counts; each dot represents an sgRNA. SSC, side scatter.