Figure 4. Inducing allele-selective gene upregulation in HEK293 cells using heterotopic enhancer activation.

a, Schematic of the human APOA4 and APOC3 genes and the two alleles of this locus present in HEK293 cells. E0 and P_A4/P_C3 indicate binding sites for gRNAs targeting the known shared enhancer and the promoters, respectively. E1-E6 indicate binding sites for gRNAs targeting the potential enhancer regions, that are expected to selectively target one allele over the other based on whether the SNP present in the SpCas9 PAMs (NGG) of these target sites maintain or disrupt the PAM. (Black bold letters indicate bases that maintain an intact PAM site and gray bold letters indicate bases that are expected to disrupt the PAM). Colored outlined boxes (yellow for allele 1, orange for allele 2) indicate PAMs targeted by E1-E6 on specific alleles, while black outlined boxes indicate PAMs targeted by E0, P_A4, P_C3 on both alleles. The SNPs in exon 2 of APOA4 and exon 3 of APOC3 that distinguish between the mRNA of the two alleles are also shown.

b-c, Total expression of APOA4/APOC3 in HEK293 cells by the bi-partite p65 aTF targeting the promoter (P_A4 /P_C3) and various sites on the enhancers including the SNP regions (E1-E6) and non-SNP region (E0) determined by RT-qPCR, normalized to HPRT1 levels, 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.

d-e, Relative quantification (percent next-generation sequencing reads of cDNA) of the two alleles of APOA4 or APOC3 mRNA when the bi-partite p65 aTF was co-expressed with a gRNA targeting the promoter (P_A4 or P_C3) alone or with one or more gRNAs targeting the known enhancer (E0) or upstream potential enhancer (E1 – E6). Open circles indicate biological replicates (n=3), bars the mean of replicates and error bars the s.e.m.