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Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing

Citation

Li, F and Wing, K and Wang, J-H and Luu, CD and Bender, JA and Chen, J and Wang, Q and Lu, Q and Nguyen Tran, MT and Young, KM and Wong, RCB and Pebay, A and Cook, AL and Hung, SSC and Liu, G-S and Hewitt, AW, Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing, Frontiers in Cellular Neuroscience, 14 Article 570917. ISSN 1662-5102 (2020) [Refereed Article]


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Copyright Statement

Copyright 2020 Li, Wing, Wang, Luu, Bender, Chen, Wang, Lu, Nguyen Tran, Young, Wong, Pébay, Cook, Hung, Liu and Hewitt. Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) https://creativecommons.org/licenses/by/4.0/

DOI: doi:10.3389/fncel.2020.570917

Abstract

CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adenoassociated 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.

Item Details

Item Type:Refereed Article
Keywords:CRISPR/Cas, CRISPR (clustered regularly interspaced short palindromic repeats), retina, retinal dystrophy, gene editing, AAV (adeno-associated virus)
Research Division:Biomedical and Clinical Sciences
Research Group:Medical biotechnology
Research Field:Gene and molecular therapy
Objective Division:Health
Objective Group:Clinical health
Objective Field:Clinical health not elsewhere classified
UTAS Author:Li, F (Dr Fan Li)
UTAS Author:Wing, K (Mr Kristof Wing)
UTAS Author:Bender, JA (Mr James Bender)
UTAS Author:Chen, J (Ms Jane Chen)
UTAS Author:Wang, Q (Miss Qi Wang)
UTAS Author:Lu, Q (Dr Qiang Lu)
UTAS Author:Nguyen Tran, MT (Mr Minh Nguyen Tran)
UTAS Author:Young, KM (Associate Professor Kaylene Young)
UTAS Author:Cook, AL (Associate Professor Tony Cook)
UTAS Author:Liu, G-S (Associate Professor Guei-Sheung Liu)
UTAS Author:Hewitt, AW (Professor Alex Hewitt)
ID Code:140830
Year Published:2020
Funding Support:National Health and Medical Research Council (1185600)
Deposited By:Menzies Institute for Medical Research
Deposited On:2020-09-10
Last Modified:2021-04-07
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