Abstract
Purpose :
Previous research showed that the removal of exon 13 of USH2A creates a functional in-frame protein, which supports the use of exon skipping as a treatment modality for USH2A-associated disease. Besides exon 13, there are 24 more in-frame exons in the USH2A gene. Here we propose to examine the possibility of expanding the exon-skipping approach to other USH2A exons with a higher load of frame-disruptive mutations.
Methods :
We used dual Cas9-gRNA pairs to delete the exons of interest in HEK293 cells. For each exon, we tested up to 10 pairs of SaCas9-sgRNAs targeting the flanking introns. We also generated several exon-deleted and exon-humanized mouse models, in which the corresponding mouse Ush2a exons were deleted or replaced with a wild-type or mutant human exon and its flanking introns. These mouse lines are used to assess the biological function of exon-excised USH2A protein and to evaluate the efficiency of selected top dual gRNA pairs in vivo.
Results :
For each exon, we identified 1-4 pairs of SaCas9-sgRNAs that achieved an exon-skipping rate of 30%-60% in cultured cells. The two double-strand breaks (DSBs) in the two flanking introns were repaired through the non-homologous end joining (NHEJ) pathway with small deletions near the target sites. We also tested dual-gRNAs exon-deletion efficiency in mouse embryos through embryonic injection and achieved an exon-deletion efficiency of 40%-70%. During the generation of exon-deletion or human exon replacement mouse models, up to 70% of founder mice from 4 different lines showed deleted or replaced exons in their genomes.
Conclusions :
We identified guide RNA pairs that can achieve highly efficient deletion of several USH2A in-frame exons in both cultured human cells and mouse embryos. Evaluation of exon-skipped USH2A protein function and the editing efficiency of the top paired gRNA candidates in mouse retinas are ongoing.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.