Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.

Abstract

CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor β-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner.

• Authors: Kathleen A Christie, Louise J Robertson, Caroline Conway, Kevin Blighe, Larry A DeDionisio, Connie Chao-Shern, Amanda M Kowalczyk, John Marshall, Doug Turnbull, M Andrew Nesbit, C B Tara Moore
• Journal: Molecular therapy : the journal of the American Society of Gene Therapy (Mol Ther) Vol. 28 Issue 8 Pg. 1846-1857 (08 05 2020) ISSN: 1525-0024 [Electronic] United States
• PMID: 32416058 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Chemical References

• RNA, Guide
• TGFB1 protein, human
• Transforming Growth Factor beta1

Topics

• Alleles
• Amino Acid Sequence
• Amino Acid Substitution
• CRISPR-Cas Systems
• Cell Line
• Gene Editing
• Genes, Dominant
• Genetic Diseases, Inborn (genetics, therapy)
• Genetic Therapy
• Genomics (methods)
• Haplotypes
• Humans
• Mutation
• Polymorphism, Single Nucleotide
• Precision Medicine
• RNA, Guide, Kinetoplastida
• Transforming Growth Factor beta1 (genetics)