Germline CRISPR/Cas9-mediated gene editing prevents vision loss in a novel mousemodel of Aniridia.
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the Sey allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy in vitro then tested it in vivo in the germline of our new mouse to validate the causality of the Sey mutation. The genomic manipulations were analyzed by PCR, as well as by Sanger and next-generation sequencing. The mice were studied by slit lamp imaging, immunohistochemistry, and western blot analyses. We successfully achieved both in vitro and in vivo germline correction of the Sey mutation, with the former resulting in an average 34.8% ± 4.6% SD correction, and the latter in restoration of 3xFLAG-tagged PAX6 expression and normal eyes. Hence, in this study we have created a novel mouse model for aniridia, demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia.
Date Issued
2020-06-12
Date Acceptance
2020-03-09
Citation
Molecular Therapy - Methods and Clinical Development, 2020, 17, pp.478-490
ISSN
2329-0501
Publisher
Elsevier (Cell Press)
Start Page
478
End Page
490
Journal / Book Title
Molecular Therapy - Methods and Clinical Development
Volume
17
Copyright Statement
© 2020 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/32258211
PII: S2329-0501(20)30032-2
Publication Status
Published online
Coverage Spatial
United States
Date Publish Online
2020-03-14