A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing

Abstract

Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype–genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.

A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System

“…The CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas)) system has rapidly become a powerful genome editing tool given its highly precise and efficient targeting method and relatively simple implementation. This molecular genetic system is remarkably powerful for modelling genetic disorders in vitro via the generation of isogenic hPSC clones that differ from the WT hPSCs in only the gene of interest [ 17 , 18 ]. In our previous work, we used CRISPR/Cas9 to generate SH3PXD2b knock out cancer cell lines [ 19 ].…”

Aberrant Splicing of INS Impairs Beta-Cell Differentiation and Proliferation by ER Stress in the Isogenic iPSC Model of Neonatal Diabetes

“…The combined use of iPSCs and CRISPR/Cas9 DNA editing technology can reduce heterogeneity by generating isogenic cell lines that share the same genetic background and differ only in a genetic variant of interest. A number of studies that utilized this approach have been recently published to study neurodegenerative [ 16 ], metabolic [ 17 ], immunological [ 18 ], and endocrine [ 19 ] diseases. Recent studies utilizing CRISPR/Cas9 and iPSC approaches revealed some pathogenic mechanisms of neonatal diabetes [ 10 , 11 ], which are particularly connected with the insulin gene mutations previously studied in mice.…”