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. Author manuscript; available in PMC: 2022 Feb 5.
Published in final edited form as: Nat Methods. 2021 Aug 5;18(9):1075–1081. doi: 10.1038/s41592-021-01224-1

Extended Data Fig. 6. Allele-selective upregulation of HBB genes in U2OS and K562 cells using heterotopic activation of enhancer.

Extended Data Fig. 6

a, Schematic of HBB gene and the three alleles present in U2OS cells. P indicates the binding site for the gRNA targeting the HBB promoter. E1-E4 indicate binding sites for gRNAs in the HS4 putative enhancer region, which are expected to target either all alleles (E1), selectively target one allele (E2, E3) or two alleles (E4) based on the PAM of the target site (black bold indicates a base that maintains an intact PAM site and gray bold indicates a base that is expected to disrupt the PAM). A SNP in exon 1 of HBB distinguishes between allele 1 (light purple) and allele 2/3 (pink).

b, Total expression of HBB in U2OS cells when the bi-partite p65 aTF was co-expressed with a gRNA targeting the promoter (P) and/or with one or more gRNAs targeting the HS4 enhancer region (E1-E4) was determined by RT-qPCR, normalized to HPRT1 levels, and calculated relative to sample with non-targeting gRNA (None). Open circles indicate biological replicates (n=3), bars the mean of replicates and error bars the s.e.m.

c, Relative quantification (percent next-generation sequencing reads of cDNA) of the three alleles of HBB mRNA when the bi-partite p65 aTF was co-expressed with a gRNA targeting the promoter (P) alone or with one or more gRNAs targeting the HS4 enhancer region (E1-E4). Open circles indicate biological replicates (n=3), bars the mean of replicates and error bars the s.e.m.