Walker, AJ;
Graham, C;
Greenwood, M;
Woodall, M;
Maeshima, R;
O'Hara-Wright, M;
Sanz, DJ;
Guerrini, I;
Aldossary, AM;
O'Callaghan, C;
et al.
Walker, AJ; Graham, C; Greenwood, M; Woodall, M; Maeshima, R; O'Hara-Wright, M; Sanz, DJ; Guerrini, I; Aldossary, AM; O'Callaghan, C; Baines, DL; Harrison, PT; Hart, SL
(2023)
Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing.
Mol Ther Methods Clin Dev, 31.
p. 101140.
ISSN 2329-0501
https://doi.org/10.1016/j.omtm.2023.101140
SGUL Authors: Baines, Deborah
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Abstract
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.
Item Type: | Article | ||||||||||||||||||
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Additional Information: | © 2023 The Author(s). This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). | ||||||||||||||||||
Keywords: | CRISPR-Cas9, cystic fibrosis, nanoparticles, splice mutation, targeted excision | ||||||||||||||||||
SGUL Research Institute / Research Centre: | Academic Structure > Infection and Immunity Research Institute (INII) | ||||||||||||||||||
Journal or Publication Title: | Mol Ther Methods Clin Dev | ||||||||||||||||||
ISSN: | 2329-0501 | ||||||||||||||||||
Language: | eng | ||||||||||||||||||
Dates: |
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Publisher License: | Creative Commons: Attribution 4.0 | ||||||||||||||||||
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PubMed ID: | 38027060 | ||||||||||||||||||
Web of Science ID: | WOS:001112148100001 | ||||||||||||||||||
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URI: | https://openaccess.sgul.ac.uk/id/eprint/116011 | ||||||||||||||||||
Publisher's version: | https://doi.org/10.1016/j.omtm.2023.101140 |
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