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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acidaminococcus / genetics*
  • Alleles
  • Bacterial Proteins / genetics
  • Biopsy
  • CRISPR-Associated Proteins / genetics*
  • Cell Culture Techniques
  • Cell Line
  • Cystic Fibrosis / genetics
  • Cystic Fibrosis / pathology
  • Cystic Fibrosis / therapy*
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics*
  • Endonucleases / genetics
  • Gene Editing / methods*
  • Humans
  • Intestines / pathology
  • Organoids
  • Point Mutation
  • RNA Splice Sites / genetics
  • RNA Splicing / genetics

Substances

  • Bacterial Proteins
  • CFTR protein, human
  • CRISPR-Associated Proteins
  • RNA Splice Sites
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Endonucleases