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

Abstract

Purpose : Aniridia is a rare congenital eye disorder, which occurs due to mutations in the paired box gene 6 (PAX6), and results in vision loss. Currently, there is no cure or long-term vision-saving therapy for aniridia. One possible approach to treating aniridia is CRISPR-mediated gene editing therapy. We hypothesize that CRISPR-mediated gene editing can increase the expression of PAX6, improve the structure and function of the eye, and ultimately rescue the mutant phenotype.

Methods : We carried out in vitro optimization of several CRISPR strategies in Pax6^Sey/Sey mouse embryonic stem cells. Functionality of each strategy was validated by Sanger sequencing on pooled lysed cells. To improve the Sey mouse model for CRISPR-based experiments, we utilized CRISPR/Cas9 technology to insert a 3xFLAG tag on the Sey allele (Fey), this allowed the differential detection of protein from each allele. As a control we also tagged a WT Pax6 allele (Fax). The next step was to test the germline ability of our best CRISPR strategy, and validate the causality of the Pax6^Sey mutation prior to somatic therapy. The corrected mice were generated by microinjecting the CRISPR strategy into Pax6^Fey/+ and Pax6^+/+ zygotes. The genomic manipulation was analyzed by Sanger sequencing of founders. The structure of the eye in the corrected Fey mice was compared to WT control mice using slit lamp imaging, and immunohistochemistry analysis.

Results : Our best CRISPR strategy corrected the Pax6 mutation in 43% of the alleles when extracted from whole cell lysate. Furthermore, initial studies show no mutagenesis on WT allele when CRISPR components were delivered to cells by electroporation. The mutant phenotype was rescued in 2 of the 15 founders. On an allelic level, we achieved a correction rate of 25% for the Fey allele (2/8). Sequencing results also revealed indels on WT allele with only 1 phenotypically deleterious hit, a rate of 4.5% (1/22), and 3 phenotypically benign hits with a rate of 14% (3/22). Therefore, our guide RNA does not have the specificity to discriminate between WT and Sey alleles when delivered by microinjection into mouse zygotes.

Conclusions : We have developed a preliminary CRISPR strategy that corrected the Pax6^Sey mutation in both cells and mouse zygotes. We have generated an improved mouse model for aniridia, and demonstrated that germline correction of the Pax6^Sey mutation alone rescues the mutant phenotype.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.