1. Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.
- Author
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Christie KA, Robertson LJ, Conway C, Blighe K, DeDionisio LA, Chao-Shern C, Kowalczyk AM, Marshall J, Turnbull D, Nesbit MA, and Moore CBT
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Cell Line, Genomics methods, Haplotypes, Humans, Polymorphism, Single Nucleotide, Precision Medicine, RNA, Guide, CRISPR-Cas Systems, Transforming Growth Factor beta1 genetics, Alleles, CRISPR-Cas Systems, Gene Editing, Genes, Dominant, Genetic Diseases, Inborn genetics, Genetic Diseases, Inborn therapy, Genetic Therapy, Mutation
- 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., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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