Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing
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Abstract
ABSTRACT CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo ; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo . We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and sgRNA targeting YFP and a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre::Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vitro and in vivo .
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AAV2.7m8 is a useful delivery vector for cells across retinal cell types.
“Overall, our results indicated AAV2.7m8 to be a useful delivery vector for cells across retinal cell types, confirming several previous studies (29–32).”
From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [30]
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Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina
Kumar, S., Hsiao, Y.-W., Wong, V. H. Y., Aubin, D., Wang, J.-H., Lisowski, L., Rakoczy, E. P., Li, F., Alarcon-Martinez, L., Gonzalez-Cordero, A., et al. (2024) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2408345121 · Cited by 19 · see citing works
Self-citationSiles, L., Ruiz-Nogales, S., Méndez-Vendrell, P., & Pomares, E. (2026) · Cells · doi:10.3390/cells15050457
- Authors
- Li, F., Wing, K., Wang, J.-H., Luu, C. D., Bender, J. A., Chen, J., Wang, Q., Lu, Q., Nguyen Tran, M. T., Young, K. M., Wong, R. C. B., Pébay, A., Cook, A. L., Hung, S. S. C., Liu, G.-S., & Hewitt, A. W.
- Affiliations
- University of Tasmania (AU)
- The Royal Victorian Eye & Ear Hospital (AU)
- The University of Melbourne (AU)
- The Royal Melbourne Hospital (AU)
- Corresponding author
- Liu, Guei-Sheung
- Hewitt, Alex W.
- ORCID
- Venue
- Frontiers in Cellular Neuroscience
- Publisher
- Frontiers Media SA
- Published
- 2020-09-10
- Volume
- 14
- Pages
- 570917-570917
- ISSN
- 1662-5102 (ISSN-L)
- PMC
- PMC7511709
- OpenAlex
- W3084652398
- Cited by
- 34
- References
- 4
- Citations by year
- 2026: 2 · 2025: 4 · 2024: 12 · 2023: 8 · 2022: 3 · 2021: 4 · 2020: 1
- FWCI
- 1.38
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Cellular Neuroscience (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UTAS Research Repository (acceptedVersion)
- UTAS Research Repository (submittedVersion)
- Minerva Access (University of Melbourne) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- National Health and Medical Research Council
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- adeno-associated virus delivery, SpCas9, SaCas9, CRISPR/Cas12a, intravitreal injection, inherited retinal disease
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