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

Kathleen A Christie,David G Courtney,M Andrew Nesbit,Connie Chao Shern,C B Tara Moore,Laura C Mairs,Larry A Dedionisio,Shyamasree De Majumdar

doi:10.1038/s41598-017-16279-4

Kathleen A Christie, David G Courtney + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-16279-4

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Journal: Scientific Reports	Publication Date: Nov 23, 2017
Citations: 68	License type: open-access

Affiliation: University of Ulster

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.

Highlights

The promise of personalised gene therapy has been brought nearer fruition with the recent advances in the field of genome engineering, the development of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) systems
Dominant negative disorders that are the result of an accumulation of mutant protein can be targeted by allele-specific CRISPR mediated gene disruption via non-homologous end-joining (NHEJ)
We have shown in vivo that gene disruption via NHEJ offers a viable approach to achieve gene silencing

Summary

Introduction

The promise of personalised gene therapy has been brought nearer fruition with the recent advances in the field of genome engineering, the development of Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR associated protein (Cas) systems. Allele-specific gene disruption via NHEJ is a potential approach to treat dominant negative disorders, in which the causative gene is haplosufficient; this involves targeting the mutant allele alone for disruption, leaving the wild-type allele intact and restoring the phenotype[6,7,8,9]. CRISPR/Cas[9] holds immense promise, one caveat to the use of the system is that Cas[9] nuclease has been shown to tolerate mismatches between the guide sequence and the target[10,11] This can lead to off-targeting elsewhere in the genome or, in this case, cleavage of the wild-type allele. Corneal dystrophies linked to these 9 genes predominantly result from missense mutations or small in-frame insertions or deletions that cause disease by a dominant negative effect of the mutant protein[17]

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Conclusion