Genome writing and Targeted Delivery of the <i>NKX6-3/ANK1</i> gene cluster and its Type 2 Diabetes GWAS Variants to Human iPSCs

Genome-wide association studies (GWAS) identified over 600 loci containing single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes (T2D), most of which reside in non-coding regions. Among the set of T2D SNPs, linking causal genome variants to disease risk experimentally has remained a challenge; however, advances in synthetic mammalian genome writing techniques now enable the delivery of multiple haplotypes to human induced pluripotent stem cells (hiPSCs) to create a series of isogenic cell lines that can be differentiated and phenotyped <i>in vitro</i>. Here, to begin efforts in dissecting a T2D GWAS locus, we engineered an <i>NKX6-3/ANK1</i> gene cluster knockout hiPSC line and introduced a landing pad facilitating the delivery of synthetic haplotype payloads. We built four haplotypes, including several that are not observed in nature, containing risk SNPs spanning the <i>NKX6-3/ANK1</i> gene cluster using a method called "variant Switching Auxotrophic markers for Integration" (vSwAP-In), and integrated them precisely into hiPSCs. <i>NKX6-3/ANK1</i> deletion blocked pancreatic progenitor and skeletal muscle differentiation, suggesting that <i>NKX6-3</i> and <i>ANK1</i> are required for early pancreatic and skeletal muscle development, and perhaps related to the existence of two nonoverlapping sets of SNPs in linkage disequilibrium that associate with the expression of the two adjacent genes. When <i>NKX6-3</i>/<i>ANK1</i> T2D "Risk" haplotypes were reintroduced, skeletal muscle and pancreatic progenitor differentiation capabilities were restored. <i>ANK1</i> expression was elevated in the <i>ANK1</i> Risk and All-Risk haplotypes compared to the <i>NKX6-3</i> Risk and Non-Risk haplotypes, establishing a functional experimental platform to examine risk SNP clusters in their native contexts. Overall, this work establishes a platform for the dissection of GWAS loci using synthetic haplotype genomics in hiPSCs.