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.
Highlights
Cystic fibrosis (CF) is a chronic and progressive disorder affecting more than 70,000 people worldwide, characterised by dysfunctional secretory epithelial cells which cause obstructions in the lung airways and pancreatic ducts [1]
For each of the three mutations, we showed that CRISPR Cas9/gRNA pairs could be used to successfully excise them via a non-homologous end joining (NHEJ) pathway
For each of the three deep-intronic mutations, multiple gRNAs were designed either side of the CF-causing single nucleotide substitution to create a deletion of !50bp
Summary
Cystic fibrosis (CF) is a chronic and progressive disorder affecting more than 70,000 people worldwide, characterised by dysfunctional secretory epithelial cells which cause obstructions in the lung airways and pancreatic ducts [1]. The disease is caused by mutations in both alleles of the CFTR gene, which encodes an apical membrane Cl-/HCO3- channel [2,3,4]. Of the 2019 sequence variants identified in the CFTR gene to date (www.genet.sickkids.on.ca/ Home.html), at least 281 have been characterised as disease-causing [www.cftr2.org; 5], and have been stratified into five or six different classes according to disease severity and the molecular basis by which they disrupt ion channel activity [6,7]. Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations data collection and analysis, decision to publish, or preparation of the manuscript
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