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

D G Courtney, J E Moore, Sarah Atkinson, E Maurizi, E H A Allen, D M L Pedrioli, W H I McLean, M. Andrew Nesbit, Tara Moore

Research output: Contribution to journalArticle

45 Citations (Scopus)

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.
LanguageEnglish
Pages108-112
Number of pages5
JournalGene Therapy
Volume23
DOIs
Publication statusPublished - 20 Aug 2015

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
DNA Cleavage
Juvenile Epithelial of Meesmann Corneal Dystrophy
Single Nucleotide Polymorphism
Mutation
DNA End-Joining Repair
Alleles
Corneal Stroma
Inborn Genetic Diseases
Missense Mutation
Sequence Analysis
Messenger RNA
In Vitro Techniques
Therapeutics
Research
Proteins

Keywords

  • CRISPR/Cas9
  • corneal dystrophy
  • gene editing

Cite this

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 ; Moore, Tara. / CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. In: Gene Therapy. 2015 ; Vol. 23. pp. 108-112.
@article{dbc065a259214b47b0ff5fc93a538bcf,
title = "CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting",
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.",
keywords = "CRISPR/Cas9, corneal dystrophy, gene editing",
author = "Courtney, {D G} and Moore, {J E} and Sarah Atkinson and E Maurizi and Allen, {E H A} and Pedrioli, {D M L} and McLean, {W H I} and Nesbit, {M. Andrew} and Tara Moore",
year = "2015",
month = "8",
day = "20",
doi = "10.1038/gt.2015.82",
language = "English",
volume = "23",
pages = "108--112",
journal = "Gene Therapy",
issn = "0969-7128",

}

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; Moore, Tara.

In: Gene Therapy, Vol. 23, 20.08.2015, p. 108-112.

Research output: Contribution to journalArticle

TY - JOUR

T1 - CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

AU - Courtney, D G

AU - Moore, J E

AU - Atkinson, Sarah

AU - Maurizi, E

AU - Allen, E H A

AU - Pedrioli, D M L

AU - McLean, W H I

AU - Nesbit, M. Andrew

AU - Moore, Tara

PY - 2015/8/20

Y1 - 2015/8/20

N2 - 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.

AB - 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.

KW - CRISPR/Cas9

KW - corneal dystrophy

KW - gene editing

UR - https://pure.ulster.ac.uk/en/publications/crisprcas9-dna-cleavage-at-snp-derived-pam-enables-both-in-vitro--5

U2 - 10.1038/gt.2015.82

DO - 10.1038/gt.2015.82

M3 - Article

VL - 23

SP - 108

EP - 112

JO - Gene Therapy

T2 - Gene Therapy

JF - Gene Therapy

SN - 0969-7128

ER -