Engineering compact RNA-guided endonucleases for CAG/CTG repeats editing

Mangin, Antoine 2024. Engineering compact RNA-guided endonucleases for CAG/CTG repeats editing. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Expansion of CAG/CTG repeats is implicated in 15 distinct neurodegenerative or neuromuscular diseases for which there are currently no cures. Huntington's disease, characterised by autosomal dominant inheritance, is a condition triggered by CAG repeat expansion within the Huntington gene. Our laboratory has demonstrated the potential of utilising the Sp Cas9 nickase to reduce the repeats to a non-pathogenic length. However, the large size of Sp Cas9 poses challenges for packaging with its guide RNA into a single adeno-associated virus (AAV) capsid, which is considered one of the most effective delivery methods available. To address this limitation, I developed compact hybrid Cas9 variants capable of fitting within a single AAV capsid alongside their guide RNA. Additionally, I assessed newly characterised RNA-guided endonuclease nickase for their effectiveness on CAG/CTG repeats. This thesis highlights that altering the PID of smaller Cas9 does not consistently affect PAM sequence recognition. Furthermore, I have demonstrated that incorporating a linker into hybrid Cas9 constructs can enhance their expression in HEK-derived cells. Moreover, I demonstrated that the recently characterised RNA-guided endonucleases are unable to target and reduce CAG repeats, an achievement accomplished only by Sp Cas9.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Medicine
Date of First Compliant Deposit:	10 September 2024
Last Modified:	10 Sep 2024 10:35
URI:	https://orca.cardiff.ac.uk/id/eprint/171963

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