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Cited by 92CRISPR and Genetic EngineeringCystic Fibrosis Research AdvancesRNA regulation and disease

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

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Sanz, David J., Hollywood, Jennifer A., Scallan, Martina F., Harrison, Patrick T.·2017·PLOS ONE
DOI 10.1371/journal.pone.0184009PMC PMC5581164
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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.

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  • SpCas9 reversed the 3849+10kbC>T splicing defects exclusively with sgRNA pairs that deleted the intronic region containing the mutation; individual sgRNA targeting the mutation (+1, +5, +18) did not repair the splicing defect.

    “We found that, consistently with the most commonly used genome editing strategies27,51, SpCas9 reversed the splicing defects exclusively with sgRNA pairs which deleted the intronic region containing the mutation (Supplementary Fig. 11a, b); in contrast to AsCas12a, SpCas9 combined with an individual sgRNA targeting the mutation (+1, +5, +18) did not repair the splicing defect (Supplementary Fig. 1”

    From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [27]

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  • CRISPR-nucleases have been successfully used to induce NHEJ to knockout genes or genomic regulatory elements, including permanent repair of splicing defects in CFTR minigene models.

    “CRISPR-nucleases have been successfully used to induce NHEJ to knockout genes or genomic regulatory elements25,26. This includes permanent repair of splicing defects in CFTR minigene models27 as an alternative to the transient and inefficient use of oligonucleotides or spliceosome-mediated RNA trans-splicing (SMaRT) strategies28,29.”

    From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [27]

    The Results section, “Targeted excision of CF-causing mutations is highly efficient and rescues splicing defects” (page unavailable), reports that Cas9/gRNA-mediated deletion rescued splicing in CFTR minigene assays. The Methods section, “Minigene assay” (page unavailable), says transcripts were analysed 48 hours after transfection; the paper does not establish that the splicing repair was permanent. The gene-knockout statement and comparison with oligonucleotide or SMaRT strategies carry other citations, so they are not judged against this source. (LLM, conf=0.82)

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  • Delivery of CRISPR-SpCas9 with multiple sgRNAs was shown to correct CFTR intronic splicing mutation in cellular minigene models by inducing deletions, although quite large in size (around 150-200 bp), of the intronic mutation and surrounding sequences.

    “However, the delivery of CRISPR-SpCas9 with multiple sgRNAs was shown to correct CFTR intronic splicing mutation in cellular minigene models by inducing deletions, although quite large in size (around 150–200 bp), of the intronic mutation and surrounding sequences27.”

    From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [27]

    The Materials and methods (Minigene assay; page unavailable) describe transfecting mutant CFTR minigenes in HEK293T cells with a plasmid expressing Cas9 and two gRNAs. The Abstract and Results report targeted excision and restored splicing. However, the Results (Design and screening of gRNA pairs; page unavailable) specify deletions of at least 50 bp, not the claimed size of around 150–200 bp. (LLM, conf=0.86)

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Authors
Sanz, D. J., Hollywood, J. A., Scallan, M. F., & Harrison, P. T.
Affiliations
University College Cork (IE)
Corresponding author
Harrison, Patrick T.
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2017-09-01
Volume
12
Issue
9
Pages
e0184009
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0184009
PMC
PMC5581164
OpenAlex
W2753061054
Cited by
92
References
59
Citations by year
2026: 4 · 2025: 7 · 2024: 7 · 2023: 8 · 2022: 14 · 2021: 7 · 2020: 16 · 2019: 13 · 2018: 14 · 2017: 2
FWCI
5.03
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Arrow@dit (Dublin Institute of Technology) (acceptedVersion)
  • Cork Open Research Archive (University College Cork, Ireland) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
  • ResearchSpace (University of Auckland) (submittedVersion, cc-by)
Funders
  • Cystic Fibrosis Trust (GB) (VIA011)
  • Cystic Fibrosis Foundation Therapeutics (HARRIS14XX0)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Cystic Fibrosis Research Advances
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
cystic fibrosis, CFTR, CRISPR/Cas9, aberrant splicing, cryptic splice site, pseudoexon, non-homologous end joining, homology-directed repair, genome editing, 5' splice site mutation, 3' splice site mutation, transcriptional regulatory motifs
MeSH terms
RNA, Guide, CRISPR-Cas Systems, Cell Separation, Codon, Cystic Fibrosis, Exons, Flow Cytometry, Humans, Introns, Mutation, Oligonucleotides, RNA, Messenger, Gene Deletion, RNA, Guide, Kinetoplastida, Alternative Splicing, Cystic Fibrosis Transmembrane Conductance Regulator, Amino Acid Motifs, CRISPR-Cas Systems

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