Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia.

Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 () 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 small eye () mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy then tested it in the germline of our new mouse to validate the causality of the 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 and germline correction of the 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 mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia.