Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA

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dc.contributor.author Sanz, David J.
dc.contributor.author Hollywood, Jennifer A.
dc.contributor.author Scallan, Martina F.
dc.contributor.author Harrison, Patrick T.
dc.date.accessioned 2017-09-26T11:39:24Z
dc.date.available 2017-09-26T11:39:24Z
dc.date.issued 2017
dc.identifier.citation Sanz, D. J., Hollywood, J. A., Scallan, M. F. and Harrison, P. T. (2017) 'Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA', PLOS ONE, 12(9), e0184009 (13pp). doi: 10.1371/journal.pone.0184009 en
dc.identifier.volume 12
dc.identifier.issued 9
dc.identifier.issn 1932-6203
dc.identifier.uri http://hdl.handle.net/10468/4812
dc.identifier.doi 10.1371/journal.pone.0184009
dc.description.abstract Cystic Fibrosis is an autosomal recessive disorder caused by mutations in the CFTR gene. CRISPR mediated, template-dependent homology-directed gene editing has been used to correct the most common mutation, c.1521_1523delCTT / p.Phe508del (F508del) which affects similar to 70% of individuals, but the efficiency was relatively low. Here, we describe a high efficiency strategy for editing of three different rare CFTR mutations which together account for about 3% of individuals with Cystic Fibrosis. The mutations cause aberrant splicing of CFTR mRNA due to the creation of cryptic splice signals that result in the formation of pseudoexons containing premature stop codons c.1679+1634A>G (1811+1.6kbA>G) and c.3718-2477C>T (3849+10kbC>T), or an out-of-frame 5' extension to an existing exon c.3140-26A>G (3272-26A>G). We designed pairs of Cas9 guide RNAs to create targeted double-stranded breaks in CFTR either side of each mutation which resulted in high efficiency excision of the target genomic regions via non-homologous end-joining repair. When evaluated in a mini-gene splicing assay, we showed that targeted excision restored normal splicing for all three mutations. This approach could be used to correct aberrant splicing signals or remove disruptive transcription regulatory motifs caused by deep-intronic mutations in a range of other genetic disorders. en
dc.description.sponsorship Cystic Fibrosis Foundation Therapeutics (HARRIS14XX0); Cystic Fibrosis Trust (VIA011) en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher Public Library of Science en
dc.relation.uri http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184009
dc.rights © 2017, Sanz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. en
dc.rights.uri https://creativecommons.org/licenses/by/4.0/
dc.subject Mutation en
dc.subject Polymerase chain reaction en
dc.subject Introns en
dc.subject Reverse transcriptase-polymerase chain reaction en
dc.subject Plasmid construction en
dc.subject Sequence analysis en
dc.subject Deletion mutation en
dc.subject Non-homologous end en
dc.title Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA en
dc.type Article (peer-reviewed) en
dc.internal.authorcontactother Patrick Harrison, Physiology, University College Cork, Cork, Ireland. +353-21-490-3000 Email: p.harrison@ucc.ie en
dc.internal.availability Full text available en
dc.description.version Published Version en
dc.internal.wokid WOS:000408816900016
dc.contributor.funder Cystic Fibrosis Foundation
dc.contributor.funder Cystic Fibrosis Foundation Therapeutics
dc.contributor.funder Cystic Fibrosis Trust
dc.description.status Peer reviewed en
dc.identifier.journaltitle PLoS ONE en
dc.internal.IRISemailaddress p.harrison@ucc.ie en
dc.identifier.articleid e0184009


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© 2017, Sanz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Except where otherwise noted, this item's license is described as © 2017, Sanz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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