Regulation of γ-globin expression by the BGLT3 locus and transcript. (A) Schematic diagram of BGLT3 deletion (dotted rectangle) generated by CRISPR/Cas9 with gRNAs (black arrows) targeting the 5′ and 3′ ends of the BGLT3 gene. Blue arrow indicates position of gRNA used for CRISPRi. Red arrow indicates position of ASO used to reduce BGLT3 transcript. (B) Expression of BGLT3 and γ-globin in homozygous BGLT3 deletion clones 2 and 30 and control cells (ctrol; no gRNA) determined by qRT-PCR. The results were normalized to actin. (C) Expression of ^Aγ- and ^Gγ-globin determined with gene-specific intronic primers for BGLT3-deleted clone 2 compared with control cells. (D) Expression of BGLT3 and γ-globin monitored by qRT-PCR after transfection of CD34⁺ cells with an ASO directed against BGLT3 or control oligonucleotide. (E) Transcription of erythroid marker genes monitored by qRT-PCR after transfection of CD34⁺ cells with a BGLT3 ASO or control. (F) ^Aγ- and ^Gγ-globin expression using gene-specific intron probes after transfection of CD34⁺ cells with a BGLT3 ASO or control. (G) ChiP-qPCR was performed with an HA antibody and primers within BGLT3 and at nontargeted control loci 3′ HS1 and the γ-globin promoter. (H) Expression of BGLT3 after CRISPRi and for control cells (no gRNA). (I) ^Aγ- and ^Gγ-globin expression using gene-specific intron probes in dCas9 cells and dCas9/BGLT3 gRNA cells. Error bars indicate standard deviation; n = 3 biological replicates. Primer sequences listed in supplemental Table 1; antibodies listed in supplemental Table 2. **P < .01 by Student t test. IgG, immunoglobulin G.