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Both hard copy thesis and e-thesis Entire Thesis Restricted Not applicable This thesis is due for publication or the author is actively seeking to publish this material Harrison, Patrick Scallan, Martina Hollywood, Jennifer A. Physiology, College of Medicine and Health, University College Cork 2014-02-24T15:00:06Z 2015-02-25T05:00:06Z 2013 2013 Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a CF cell line. Firstly, the most common mutation, ΔF508, was corrected using a pair of existing ZFNs, which cleave in intron 9, and the donor repair plasmid pITR-donor-XC, which contains the correct CTT sequence and two unique restriction sites. HDR was initially determined to be <1% but further analysis by next generation sequencing (NGS) revealed HDR occurred at a level of 2%. This relatively low level of repair was determined to be a consequence of distance from the cut site to the mutation and so rather than designing a new pair of ZFNs, the position of the existing intron 9 ZFNs was exploited and attempts made to correct >80% of CF-causing mutations. The ZFN cut site was used as the site for HDR of a mini-gene construct comprising exons 10-24 from CFTR cDNA (with appropriate splice acceptor and poly A sites) to allow production of full length corrected CFTR mRNA. Finally, the ability to cleave closer to the mutation and mediate repair of CFTR using the latest gene editing tool CRISPR/Cas9 was explored. Two CRISPR gRNAs were tested; CRISPR ex10 was shown to cleave at an efficiency of 15% and CRISPR in9 cleaved at 3%. Both CRISPR gRNAs mediated HDR with appropriate donor plasmids at a rate of ~1% as determined by NGS. This is the first evidence of CRISPR induced HDR in CF cell lines. Not peer reviewed Accepted Version application/pdf Hollywood, J. 2013. Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools. PhD Thesis, University College Cork. 177 https://hdl.handle.net/10468/1407 en University College Cork © 2013, Jennifer Hollywood http://creativecommons.org/licenses/by-nc-nd/3.0/ CFTR ZFN CRISPR/Cas9 Gene editing Cystic fibrosis gene Cystic fibrosis--Gene therapy RNA editing true Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools Doctoral thesis Doctoral PhD (Medicine and Health)info:eu-repo/semantics/openAccess