Biology subjects

Cipriani, F.

Publications and source records attributed to Cipriani, F..

3 recordsLinked to original sources

bioRxiv2026

Genome writing and Targeted Delivery of the NKX6-3/ANK1 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 in vitro. Here, to begin efforts in dissecting a T2D GWAS locus, we engineered an NKX6-3/ANK1 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 NKX6-3/ANK1 gene cluster using a method called "variant Switching Auxotrophic markers for Integration" (vSwAP-In), and integrated them precisely into hiPSCs. NKX6-3/ANK1 deletion blocked pancreatic progenitor and skeletal muscle differentiation, suggesting that NKX6-3 and ANK1 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 NKX6-3/ANK1 T2D "Risk" haplotypes were reintroduced, skeletal muscle and pancreatic progenitor differentiation capabilities were restored. ANK1 expression was elevated in the ANK1 Risk and All-Risk haplotypes compared to the NKX6-3 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. Significance StatementGenome-wide association studies have been used to identify disease-associated SNPs; however, most SNPs lie in non-coding regions, making functional experimentation difficult to perform. Using vSwAP-In, a yeast-based DNA variant-building method, and mSwAP-In, a mammalian genome engineering approach, we establish a platform for functional GWAS dissection in hiPSCs. This platform allows us to build DNA harboring virtually any combination of disease-risk SNPs, allowing for functional characterization of SNPs without the limitations of linkage disequilibrium. We demonstrate this approach using a Type 2 diabetes GWAS gene cluster, NKX6-3/ANK1.

genetics↗

bioRxiv2025

Genetics of growth rate in induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) have transformed biomedical research by enabling the generation of diverse cell types from accessible somatic tissues. However, certain fundamental biological properties, such as the genetic and epigenetic determinants of iPSC proliferation, remain poorly characterized. We measured the growth of iPSC lines derived from 602 unique donors using high-throughput time-lapse imaging, quantified proliferation through a growth Area-Under-the-Curve (gAUC) phenotype, and correlated gAUC with the gene expression and genotype of the cell lines. We identified 3,091 genes associated with gAUC, many of which are well established regulators of cell proliferation. We also found that rare deleterious variants in WDR54 were associated with reduced iPSC growth and that WDR54 was differentially expressed with respect to gAUC. Although no common variants showed a genome-wide association with gAUC, iPSC lines from monozygotic twins were highly correlated, and common genetic variation explained approximately 71-75% of the variance in iPSC growth rates. These results indicate a complex genetic architecture of iPSC growth rates, where rare, large-effect variants in important growth regulators, including WDR54, are layered onto a highly polygenic background. These findings have important implications for the design of pooled iPSC-based studies and disease models, which may be confounded by intrinsic growth differences.

developmental biology↗

bioRxiv2024

EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control

Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. With EasyGrid, we optimized grid preparation for different purified macromolecular complexes and subsequently determined their structure with cryo-EM. We also demonstrated how the platform allows better vitrification of large, mammalian cells compared to standard plunge-freezing. Automated sample preparation with EasyGrid establishes an advanced, high-throughput platform for both single-particle cryo-EM and cellular cryo-ET sample preparation.

molecular biology↗