# CRISPR/Cas-mediated gene editing of retinal cells in vivo

## TL;DR

This study shows that CRISPR/Cas can be used to edit genes in retinal cells of mice using a virus, with minimal impact on retinal function.

## Contribution

The study demonstrates effective in vivo CRISPR/Cas gene editing in retinal cells using AAV2 delivery, with quantifiable results in a transgenic mouse model.

## Key findings

- AAV2-mediated delivery of CRISPR/Cas significantly reduced YFP-positive retinal cells by 84%.
- Electroretinography showed no significant change in retinal function after gene editing.
- The Thy1-YFP mouse model proved effective for assessing retinal gene modification.

## Abstract

CRISPR/Cas has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt Yellow Fluorescent Protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilising the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for genome modification of retinal cells in vivo.

sgRNA plasmids were designed to target YFP and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver SpCas9 and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts.

AAV2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP, significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% CI: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes.

Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral mediated delivery of CRISPR/Cas.