CRISPR/Cas9-mediated knock-in of V5-tag in human iPS cells : Developing a tool for studying the function and localization of ERCC6L2

Abstract

Bone marrow failure leads to decreased production of blood cells. Inherited bone marrow failure can develop as a consequence of germline mutations, which can increase the risk of developing malignant disorders, such as myelodysplastic syndrome and acute myeloid leukemia. In the past decade, biallelic germline mutations in ERCC6L2 gene have been shown to cause bone marrow failure. Additionally, these mutations increase the risk of accumulating somatic TP53 mutations and the development of a poor prognosis subtype of acute myeloid leukemia, known as erythroleukemia (AML M6). ERCC6L2 protein functions in DNA repair mechanisms. However, the function of the mutated ERCC6L2 protein has not yet been fully investigated. Mutations in ERCC6L2 may cause premature termination of translation, potentially leading to a truncated protein product, which may alter its function.

The aim of this study was to develop a tool that could be used in future research to investigate the product, function, and localization of the truncated ERCC6L2 protein. The study utilized human induced pluripotent stem cells (hiPSCs) generated from fibroblasts of patients carrying ERCC6L2 germline mutations. This study employed the CRISPR/Cas9 gene editing technique by inserting a V5 epitope tag into ERCC6L2, which can be detected using an appropriate antibody. Successful gene editing was screened using agarose gel electrophoresis of PCR amplified samples and confirmed by Sanger sequencing.

The results demonstrated that the V5 epitope tag was successfully inserted into ERCC6L2. The goal was to produce cell lines that would represent with homozygous insertion of the V5 epitope tag. In addition to homozygous cell lines, heterozygous cell lines were also obtained.