Comparison of CRISPR/Cas Endonucleases forin vivoRetinal Gene Editing
AuthorLi, F; Wing, K; Wang, J-H; Luu, CD; Bender, JA; Chen, J; Wang, Q; Lu, Q; Nguyen Tran, MT; Young, KM; ...
Source TitleFrontiers in Cellular Neuroscience
PublisherFRONTIERS MEDIA SA
Centre for Eye Research Australia (CERA)
Ophthalmology (Eye & Ear Hospital)
Document TypeJournal Article
CitationsLi, 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., Pebay, A., Cook, A. L., Hung, S. S. C., Liu, G. -S. & Hewitt, A. W. (2020). Comparison of CRISPR/Cas Endonucleases forin vivoRetinal Gene Editing. FRONTIERS IN CELLULAR NEUROSCIENCE, 14, https://doi.org/10.3389/fncel.2020.570917.
Access StatusOpen Access
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 a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the 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 vivo.
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