Dataset Information

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

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 ~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.

SUBMITTER: Sanz DJ

PROVIDER: S-EPMC5581164 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Sanz David J DJ Hollywood Jennifer A JA Scallan Martina F MF Harrison Patrick T PT

PloS one 20170901 9

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 ~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 muta ...[more]

PMID: 28863137

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Project description:Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl(-) and Na(+) epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%-65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.

Dataset Information

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

Publications

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

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