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The impact of FAN1 on a CRISPR-Cas9 gene editing approach for Huntington’s Disease

Heraty, Laura 2023. The impact of FAN1 on a CRISPR-Cas9 gene editing approach for Huntington’s Disease. PhD Thesis, Cardiff University.
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Fifteen neurodegenerative or neuromuscular diseases are caused by expanded CAG/CTG repeats at multiple loci, all of which are without disease-modifying treatment. One example is Huntington’s disease (HD), an autosomal dominant disease caused by an expanded CAG repeat within exon one of the HTT gene. In HD the length of the CAG repeat tract is inversely correlated with the age at onset, however, recent genome-wide association studies have identified several genetic modifiers of disease. One such modifier was mapped to the region containing FAN1. FAN1, a DNA repair nuclease, is thought to provide protection against somatic expansion with loss-of-function variants associated with an earlier age of HD onset. Our laboratory has previously described a potential therapeutic approach for CAG/CTG diseases. This involves a CRISPR-Cas9 D10A nickase gene editing system, that is capable of contracting expanded repeats in human cell-lines, to nonpathogenic lengths. Given the central role of FAN1 in protecting against somatic expansion, the aim of this thesis was to characterise whether FAN1 modulates CRISPR-Cas9-induced contractions. This is important for not only understanding which proteins are involved in generating or preventing nickase-induced contractions but also for patient stratification to identify patients which would benefit from this therapy. This thesis demonstrates that in a HEK293 reporter cell line knocked out for FAN1, there is an increase in Cas9 nickase-induced contractions, relative to FAN1+/+ cells. This indicates that FAN1 acts to protect against the induction of contractions. Furthermore, this protective role for FAN1 requires FAN1 binding at CAG/CTG repeats, as a DNA-binding mutant identified was not capable of rescuing contractions to FAN1+/+ levels. Additional functional domains of FAN1, including the nuclease and UBZ-binding domains are also implicated. Follow-up validation of HD-patient derived iPSC lines has been described in an effort to characterise whether this protective phenotype is recapitulated iPSC-derived neurons.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 14 May 2024
Last Modified: 14 May 2024 13:24

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