Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

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dc.contributor.advisor Harrison, Patrick en
dc.contributor.advisor Scallan, Martina en
dc.contributor.author Hollywood, Jennifer
dc.date.accessioned 2014-02-24T15:00:06Z
dc.date.available 2015-02-25T05:00:06Z
dc.date.issued 2013
dc.date.submitted 2013
dc.identifier.citation 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. en
dc.identifier.endpage 177
dc.identifier.uri http://hdl.handle.net/10468/1407
dc.description.abstract 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. en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher University College Cork en
dc.rights © 2013, Jennifer Hollywood en
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/ en
dc.subject CFTR en
dc.subject ZFN en
dc.subject CRISPR/Cas9 en
dc.subject Gene editing en
dc.subject.lcsh Cystic fibrosis gene en
dc.subject.lcsh Cystic fibrosis--Gene therapy en
dc.subject.lcsh RNA editing en
dc.title Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools en
dc.type Doctoral thesis en
dc.type.qualificationlevel Doctoral en
dc.type.qualificationname PhD (Medicine and Health) en
dc.internal.availability Full text available en
dc.description.version Accepted Version
dc.contributor.funder Physiology, College of Medicine and Health, University College Cork en
dc.description.status Not peer reviewed en
dc.internal.school Microbiology en
dc.internal.school Physiology en
dc.check.reason This thesis is due for publication or the author is actively seeking to publish this material en
dc.check.opt-out Yes en
dc.thesis.opt-out true
dc.check.entireThesis Entire Thesis Restricted
dc.check.embargoformat Both hard copy thesis and e-thesis en
dc.internal.conferring Spring Conferring 2014 en


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