Peer-Reviewed Journal Details
Mandatory Fields
Sanz, DJ;Hollywood, JA;Scallan, MF;Harrison, PT
2017
September
Plos One
Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA
Validated
WOS: 42 ()
Optional Fields
CONDUCTANCE REGULATOR GENE PULMONARY-DISEASE EPITHELIAL-CELLS DELTA-F508 CFTR CAS9 PROTEIN DOUBLE-BLIND 3849+10 KB DNA IVACAFTOR VARIANTS
12
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 similar to 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.
SAN FRANCISCO
1932-6203
10.1371/journal.pone.0184009
Grant Details