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Nat Commun. 2019 Aug 7;10(1):3556. doi: 10.1038/s41467-019-11454-9.

Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing.

Author information

1
Centre for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123, Trento, Italy.
2
Department of Development and Regeneration, CF Centre, Woman and Child, KU Leuven, Herestraat 49, Leuven, 3000, Belgium.
3
Pediatric Pulmonology, Department of Pediatrics, University Hospital Leuven, Herestraat 49, Leuven, 3000, Belgium.
4
Laboratory for Molecular Virology and Drug Discovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49, Leuven, 3000, Belgium.
5
Laboratory for Molecular Virology and Drug Discovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49, Leuven, 3000, Belgium. marianne.carlon@kuleuven.be.
6
Centre for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123, Trento, Italy. gpetris@mrc-lmb.cam.ac.uk.
7
Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, CB2 0QH, UK. gpetris@mrc-lmb.cam.ac.uk.
8
Centre for Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, 38123, Trento, Italy. anna.cereseto@unitn.it.

Abstract

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272-26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

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