CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.
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.
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK.
D G
DG
Courtney
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK.|Cathedral Eye Clinic, Belfast, Northern Ireland, UK.
J E
JE
Moore
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK.
S D
SD
Atkinson
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK.
E
E
Maurizi
Centre for Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry and Nursing, University of Dundee, Dundee, Scotland, UK.
E H A
EH
Allen
Centre for Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry and Nursing, University of Dundee, Dundee, Scotland, UK.
D M L
DM
Pedrioli
Centre for Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry and Nursing, University of Dundee, Dundee, Scotland, UK.
W H I
WH
McLean
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK.
M A
MA
Nesbit
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK.
C B T
CB
Moore
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D053837:DNA Cleavage; D005260:Female; D018390:Gene Targeting; D015316:Genetic Therapy; D006579:Heterozygote; D053551:Keratin-12 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D020641:Polymorphism, Single Nucleotide
C507495:KRT12 protein, mouse; D053551:Keratin-12