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

Research output: Contribution to journalArticle

14 Citations (Scopus)

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.
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

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Alleles
Single Nucleotide Polymorphism
Corneal Epithelium
Inborn Genetic Diseases
Reporter Genes
Base Pairing
DNA Repair
Genes
Gene Editing
Plasmids
Injections
DNA

Keywords

  • CRISPR
  • Gene editing

Cite this

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
Christie, Kathleen ; Courtney, David ; De Dionisio, Larry ; Chao Shern, Connie ; De Majumdar, Shyamasree ; Mairs, Laura ; Nesbit, Andrew ; Moore, Tara. / Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders. In: Scientific Reports. 2017 ; Vol. 7, No. 1.
@article{cc5feed96ba145aa83b713c3926e1f33,
title = "Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders",
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.",
keywords = "CRISPR, Gene editing",
author = "Kathleen Christie and David Courtney and {De Dionisio}, Larry and {Chao Shern}, Connie and {De Majumdar}, Shyamasree and Laura Mairs and Andrew Nesbit and Tara Moore",
year = "2017",
month = "11",
day = "23",
doi = "10.1038/s41598-017-16279-4",
language = "English",
volume = "7",
journal = "Scientific Reports",
issn = "2045-2322",
number = "1",

}

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders. / Christie, Kathleen; Courtney, David; De Dionisio, Larry; Chao Shern, Connie; De Majumdar, Shyamasree; Mairs, Laura; Nesbit, Andrew; Moore, Tara.

In: Scientific Reports, Vol. 7, No. 1, 16174, 23.11.2017.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders

AU - Christie, Kathleen

AU - Courtney, David

AU - De Dionisio, Larry

AU - Chao Shern, Connie

AU - De Majumdar, Shyamasree

AU - Mairs, Laura

AU - Nesbit, Andrew

AU - Moore, Tara

PY - 2017/11/23

Y1 - 2017/11/23

N2 - 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.

AB - 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.

KW - CRISPR

KW - Gene editing

UR - https://www.nature.com/articles/s41598-017-16279-4

U2 - 10.1038/s41598-017-16279-4

DO - 10.1038/s41598-017-16279-4

M3 - Article

VL - 7

JO - Scientific Reports

T2 - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 16174

ER -

Christie K, Courtney D, De Dionisio L, Chao Shern C, De Majumdar S, Mairs L et al. Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders. Scientific Reports. 2017 Nov 23;7(1). 16174. https://doi.org/10.1038/s41598-017-16279-4