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.contributor.funder	Cystic Fibrosis Foundation
dc.contributor.funder	Cystic Fibrosis Foundation Therapeutics
dc.contributor.funder	Cystic Fibrosis Trust
dc.date.accessioned	2017-09-26T11:39:24Z
dc.date.available	2017-09-26T11:39:24Z
dc.date.issued	2017
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.description.status	Peer reviewed	en
dc.description.version	Published Version	en
dc.format.mimetype	application/pdf	en
dc.identifier.articleid	e0184009
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.doi	10.1371/journal.pone.0184009
dc.identifier.issn	1932-6203
dc.identifier.issued	9
dc.identifier.journaltitle	PLoS ONE	en
dc.identifier.uri	https://hdl.handle.net/10468/4812
dc.identifier.volume	12
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

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

Files

Original bundle

Collections