Ulster University Logo

CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

Courtney, D G, Moore, J E, Atkinson, Sarah, Maurizi, E, Allen, E H A, Pedrioli, D M L, McLean, W H I, Nesbit, M. Andrew and Moore, Tara (2015) CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. Gene Therapy, 23 (1). pp. 108-112. [Journal article]

[img] Other (pdf) - Accepted Version
Indefinitely restricted to Repository staff only.

774kB

URL: http://dx.doi.org/10.1038/gt.2015.82

Abstract

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann/'s epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

Item Type:Journal article
Keywords:CRISPR/Cas9, corneal dystrophy, gene editing
Faculties and Schools:Faculty of Life and Health Sciences > School of Biomedical Sciences
Faculty of Life and Health Sciences
Research Institutes and Groups:Biomedical Sciences Research Institute
Biomedical Sciences Research Institute > Genomic Medicine
Biomedical Sciences Research Institute > Stratified Medicine
ID Code:37422
Deposited By: Dr Andrew Nesbit
Deposited On:13 Apr 2017 08:58
Last Modified:17 Oct 2017 16:28

Repository Staff Only: item control page