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

Simple item page
dc.check.embargoformatBoth hard copy thesis and e-thesisen
dc.check.entireThesisEntire Thesis Restricted
dc.check.opt-outNot applicableen
dc.check.reasonThis thesis is due for publication or the author is actively seeking to publish this materialen
dc.contributor.advisorHarrison, Patricken
dc.contributor.advisorScallan, Martinaen
dc.contributor.authorHollywood, Jennifer A.
dc.contributor.funderPhysiology, College of Medicine and Health, University College Corken
dc.date.accessioned2014-02-24T15:00:06Z
dc.date.available2015-02-25T05:00:06Z
dc.date.issued2013
dc.date.submitted2013
dc.description.abstractCystic 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.description.statusNot peer revieweden
dc.description.versionAccepted Version
dc.format.mimetypeapplication/pdfen
dc.identifier.citationHollywood, 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.endpage177
dc.identifier.urihttps://hdl.handle.net/10468/1407
dc.language.isoenen
dc.publisherUniversity College Corken
dc.rights© 2013, Jennifer Hollywooden
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/en
dc.subjectCFTRen
dc.subjectZFNen
dc.subjectCRISPR/Cas9en
dc.subjectGene editingen
dc.subject.lcshCystic fibrosis geneen
dc.subject.lcshCystic fibrosis--Gene therapyen
dc.subject.lcshRNA editingen
dc.thesis.opt-outtrue
dc.titleCystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing toolsen
dc.typeDoctoral thesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD (Medicine and Health)en
Files
Original bundle
Now showing 1 - 2 of 2
Thumbnail Image
Name:
Abstract.pdf
Size:
64.45 KB
Format:
Adobe Portable Document Format
Description:
Abstract
Thumbnail Image
Name:
JH thesis.pdf
Size:
3.33 MB
Format:
Adobe Portable Document Format
Description:
Full Text E-Theses
Download
License bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
license.txt
Size:
5.62 KB
Format:
Item-specific license agreed upon to submission
Description:
Download
Collections
Doctoral Theses
College of Medicine and Health - Doctoral Theses
College of Science, Engineering and Food Science - Doctoral Theses
Microbiology - Doctoral Theses
Physiology - Doctoral Theses