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Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing

Walker, Amy J; Graham, Carina; Greenwood, Miriam; Woodall, Maximillian; Maeshima, Ruhina; O’Hara-Wright, Michelle; Sanz, David J; ... Hart, Stephen L; + view all (2023) Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing. Molecular Therapy - Methods & Clinical Development , 31 , Article 101140. 10.1016/j.omtm.2023.101140. (In press). Green open access

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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.

Type: Article
Title: Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.omtm.2023.101140
Publisher version: https://doi.org/10.1016/j.omtm.2023.101140
Language: English
Additional information: © The Author(s), 2023. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. https://creativecommons.org/licenses/by/4.0/
Keywords: cystic fibrosis, CRISPR-Cas9, nanoparticles, splice mutation, targeted excision
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Genetics and Genomic Medicine Dept
URI: https://discovery.ucl.ac.uk/id/eprint/10181707
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