Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

Kathleen Christie, David Courtney, Larry De Dionisio, Connie Chao Shern, Shyamasree De Majumdar, Laura Mairs, Andrew Nesbit, Tara Moore

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22 Citations (Scopus)
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Abstract

CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at “off-target” sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.
Original languageEnglish
Article number16174
Number of pages11
JournalScientific Reports
Volume7
Issue number1
Early online date23 Nov 2017
DOIs
Publication statusE-pub ahead of print - 23 Nov 2017

Keywords

  • CRISPR
  • Gene editing

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    Christie, K., Courtney, D., De Dionisio, L., Chao Shern, C., De Majumdar, S., Mairs, L., ... Moore, T. (2017). Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders. Scientific Reports, 7(1), [16174]. https://doi.org/10.1038/s41598-017-16279-4