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Title: Exploring the link between CHD2 mutations and double strand break repair in developing neurons
Author: Tully, Ian James
ISNI:       0000 0005 0285 6395
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2020
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Introduction: Heterozygous mutations CHD2 cause intellectual disability and refractory epilepsy. Functional studies demonstrate a deficit in DNA double strand break (DSB) repair via nonhomologous end joining (NHEJ) in CHD2 deficient cells. This project aims to investigate the impact of CHD2 mutations on the outcomes of DSB repair in developing neurons. Methods: A doxycycline inducible CRISPR-Cas9 (iCas9) construct was integrated into the AAVS1 safe harbour. A pipeline for high-throughput analysis of CRISPR experiments was designed and implemented based on nanopore sequencing. This pipeline was used to create a CHD2- deficient human induced pluripotent stem cell (hIPSC) line, which was used to investigate the effects of CHD2 on neurodifferentiation and DSB repair. The pipeline was then adapted to monitor repair outcomes of targeted DSBs in differentiating cells. Spontaneously occurring DSBs were examined using a novel next-generation sequencing technique, INDUCE-Seq, in which unrepaired DSBs are captured from permeabilised cells in-situ and sequenced in order to provide a snapshot of DSBs existing at the time of extraction in differentiating cells. Results: Cas9 induction of DSBs demonstrated changes in the repair, with an increased rate of large deletions in the CHD2-deficient cell line. No reproducible change in the pattern of smaller indels was identified. There was a significant increase of the number of DSBs captured by INDUCE-seq at D19 of differentiation in WT cells, which was not present until D40 in CHD2-deficient cells. Differences in the enrichment of DSBs at various histone markers, gene bodies and transcription start sites (TSS) were identified between CHD2-deficient cells and WT cells. Conclusions: This study demonstrates an impact on the occurrence and repair of DSBs in CHD2- deficient cell lines. Integration of RNA-Seq data and analysis of the pattern of spontaneous breakage suggests that altered DSB repair physiology could contribute towards the phenotype exhibited by patients with CHD2 mutations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)