Single‐base editing of rs12603332 on chromosome 17q21 with a cytosine base editor regulates ORMDL3 and ATF6α expression

Background Genetic association studies have demonstrated that the SNP rs12603332 located on chromosome 17q21 is highly associated with the risk of the development of asthma. Methods To determine whether SNP rs1260332 is functional in regulating levels of ORMDL3 expression, we used a Cytosine Base Editor (CBE) plasmid DNA or a CBE mRNA to edit the rs12603332 C risk allele to the T non‐risk allele in a human lymphocyte cell line (i.e., Jurkat cells) and in primary human CD4 T cells that carry the C risk alleles. Results Jurkat cells with the rs12603332 C risk allele expressed significantly higher levels of ORMDL3 mRNA, as well as the ORMDL3 regulated gene ATF6α as assessed by qPCR compared to Jurkat clones with the T non‐risk allele. In primary human CD4 T cells, we edited 90 ± 3% of the rs12603332‐C risk allele to the T non‐risk allele and observed a reduction in ORMDL3 and ATF6α expression. Bioinformatic analysis predicted that the non‐risk allele rs12603332‐T could be the central element of the E‐box binding motif (CANNTG) recognized by the E47 transcription factor. An EMSA assay confirmed the bioinformatics prediction demonstrating that a rs12603332‐T containing probe bound to the transcription factor E47 in vitro. Conclusions SNP rs12603332 is functional in regulating the expression of ORMDL3 as well as ORMDL3 regulated gene ATF6α expression. In addition, we demonstrate the use of CBE technology in functionally interrogating asthma‐associated SNPs using studies of primary human CD4 cells. SNP rs12603332 on chromosome 17q21 is highly associated with the development of asthma. Using a Cytosine Base Editor, the rs12603332 C risk allele was edited to the T non‐risk allele in primary human CD4 T cells. This change regulated the expression of ORMDL3 and ORMDL3 regulated gene ATF6α expression. Abbreviations: ATF6α, activating transcription factor α; C, cytosine; Cas9, CRISP‐associated protein 9; Chr 17q12‐21, chromosome 17q12‐21 region; ERBB2, erb‐b2 receptor tyrosine kinase 2; GRB7, growth factor receptor bound protein 7; gRNA, guide RNA; GSDMA, gasdermin A; GSDMB, gasdermin B; IKZF3, IKAROS family zinc finger 3; LRRC3C, leucine rich repeat containing 3C; ORMDL3, ORM1 like; PGAP3, post‐GPI attachment to proteins phospholipase 3; rAPOBEC1, rat apolipoprotein‐B‐editing enzyme, catalytic polypeptide‐1; SNP, single nucleotide polymorphisms; T, thymine; UGI, uracil glycosylase inhibitor; ZPBP2, zona pellucida binding protein 2