Molecular Dissections of the DNA Repair-Deficiency Disorders Using Induced Pluripotent Stem Cells and CRISPR Interference

Abstract

Recent developments of induced pluripotent stem cell (iPSC) and genome editing technologies enable us to create countless new opportunities in clinical researches for previously incurable diseases. With these new technologies, we aim to gain deeper understandings of a group of related untreatable disorders, Cockayne Syndrome, Xeroderma Pigmentosa and the ultraviolet (UV)-Sensitive Syndrome. The joint possession of these diseases is their hereditary and also insufficiency in repair systems for DNA damages. The impairment of DNA repair systems causes successive accumulation of genomic mutations that frequently, but not always, elevates the risk of cancer, UV sensitivity of skins and neuronal damages tremendously. However, despite dysfunction of the same DNA repair machinery in the diseases, there are differences in symptoms among them, and molecular mechanisms that underpin the different manifestations are poorly understood.Here, to elucidate the mechanisms, we used the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) technology. The CRISPRi utilizes catalytically dead (d)Cas9 fused with KRAB, a transcription repressor domain, to transcriptionally repress genes of interest. Mechanistically, the repression is achieved by recruiting dCas9-KRAB to a promoter region of the gene in a single guide (sg)RNA dependent manner. By employing a disease-free CRISPRi human (h)iPSC line in which dCas9-KRAB can be induced by doxycycline (Dox) treatment, we first established CRISPRi hiPSC lines where each disease-causative gene can be repressed. Then, we examined if suppressing the causative gene by Dox treatment can recapitulate disease phenotypes. We observed that, for instance, when we suppressed ERCC6, a gene responsible for Cockayne syndrome, cell proliferation of iPSCs was impaired. Additionally, production of reactive oxygen species, which involves DNA damages, was elevated in the hiPSCs regardless of UV irradiation. The similar phenotypes were observed using hiPSCs generated from a Cockayne Syndrome patient, suggesting the CRISPRi can mirror the disease phenotypes. In this meeting, I will discuss differences in phenotypes at molecular and cellular levels by suppressing the genes that are responsible for the related disorders not only in undifferentiated iPSCs but also in differentiated hiPSCs relevant to the diseases.