AAV-mediated CRISPR/cas gene editing of retinal cells in vivo
Open accessPublished version of a preprint
Abstract
Purpose Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (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 utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. Methods Single guide RNA (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 Streptococcus pyogenes Cas9 (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. Results Adeno-associated virus 2-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% confidence interval [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 CRIS…
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Co-transduction can also be achieved by intravitreal injection, which targets the retinal ganglion cells and inner retina, albeit at much lower efficiencies than by subretinal injections.
“However, co-transduction can also be achieved by intravitreal injection, which targets the retinal ganglion cells and inner retina (Hung et al., 2016), albeit at much lower efficiencies than by subretinal injections.”
From Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting (2017) · cites this paper as Hung et al., 2016
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- Authors
- Hung, S. S. C., Chrysostomou, V., Li, F., Lim, J. K. H., Wang, J.-H., Powell, J. E., Tu, L., Daniszewski, M., Lo, C., Wong, R. C., Crowston, J. G., Pébay, A., King, A. E., Bui, B. V., Liu, G.-S., & Hewitt, A. W.
- Affiliations
- The University of Melbourne (AU)
- Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia 2Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia
- The University of Queensland (AU)
- Jinan University (CN)
- Monash University (AU)
- University of Tasmania (AU)
- Corresponding author
- Hewitt, Alex W.
- ORCID
- Venue
- Investigative Opthalmology & Visual Science
- Published
- 2016-06-29
- Volume
- 57
- Issue
- 7
- Pages
- 3470
- ISSN
- 0146-0404 (ISSN-L)
- 1552-5783
- OpenAlex
- W2461468522
- Cited by
- 125
- References
- 7
- Citations by year
- 2026: 2 · 2025: 6 · 2024: 9 · 2023: 5 · 2022: 10 · 2021: 10 · 2020: 19 · 2019: 13 · 2018: 21 · 2017: 27 · 2016: 3
- FWCI
- 11.08
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Investigative Ophthalmology & Visual Science (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- eCite Digital Repository (University of Tasmania) (submittedVersion, cc-by-nc-nd)
- Figshare (submittedVersion, cc-by-nc-nd)
- Minerva Access (University of Melbourne) (submittedVersion, cc-by-nc-nd)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- CRISPR/Cas9, adeno-associated virus type 2, in vivo gene editing, retinal cells, SpCas9, single guide RNA, intravitreal injection, electroretinogram, retinal function, inherited retinal disease, viral gene delivery
- MeSH terms
- Gene Editing, Dependovirus, Animals, Bacterial Proteins, Cells, Cultured, Electroretinography, Genetic Engineering, Luminescent Proteins, Mice, Transgenic, Plasmids, Retina, Mice, Injections, Intraocular, Clustered Regularly Interspaced Short Palindromic Repeats
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