CRISPR/Cas9-based screen identified SYNCRIP, hnRNPM, PTBP1, and RBM12 as candidate HbF regulators. (A) Scatterplot displaying RBP-focused CRISPR/Cas9 screening results. Each dot represents an sgRNA. Highlighted are NT control sgRNAs and sgRNAs targeting candidate HbF regulators. (B) HUDEP2 validation results. Controls are an NT sgRNA and a BCL11A sgRNA targeting exon 2 of BCL11A. Each candidate was targeted with 2 independent sgRNAs (n = 2 biological replicates). Plotted are means ± standard deviation. Left: Percentage of F cells quantified via HbF flow cytometry. Right: qRT-PCR of HBG1/2 mRNA as percentage of total β-like globin transcripts. Results were normalized to HPRT1. ∗P < .05 by Student’s t test. (C) CD34⁺ HSPC validation result. Controls are NT and a BCL11A sgRNA targeting the +58 erythroid enhancer of BCL11A. Each candidate was targeted with 2 independent sgRNAs (n = 3 biological replicates). Plotted are means ± standard deviation. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 by Student’s t test. Left: Percentage of HbF⁺ cells quantified via HbF flow cytometry. Center: qRT-PCR of HBG1/2 mRNA as percentage of total β-like globin transcripts. Results were normalized to HPRT1. Right: Percentage of HbF relative to NT quantified via high-performance liquid chromatography.