Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases.

Abstract

With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.

• Authors: Thomas Kocher, Patricia Peking, Alfred Klausegger, Eva Maria Murauer, Josefina Piñón Hofbauer, Verena Wally, Thomas Lettner, Stefan Hainzl, Michael Ablinger, Johann Wolfgang Bauer, Julia Reichelt, Ulrich Koller
• Journal: Molecular therapy : the journal of the American Society of Gene Therapy (Mol Ther) Vol. 25 Issue 11 Pg. 2585-2598 (11 01 2017) ISSN: 1525-0024 [Electronic] United States
• PMID: 28888469 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Chemical References

• KRT14 protein, human
• Keratin-14
• RNA, Guide
• Deoxyribonuclease I

Topics

• Base Sequence
• CRISPR-Cas Systems
• Cells, Cultured
• Deoxyribonuclease I (genetics, metabolism)
• Epidermolysis Bullosa Simplex (genetics, metabolism, pathology, therapy)
• Exons
• Gene Editing (methods)
• Gene Expression
• High-Throughput Nucleotide Sequencing
• Humans
• Introns
• Keratin-14 (genetics, metabolism)
• Keratinocytes (metabolism, pathology, transplantation)
• Molecular Targeted Therapy
• Mutation
• Plasmids (chemistry, metabolism)
• RNA, Guide, Kinetoplastida (genetics)
• Recombinational DNA Repair