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

Li, F ORCID: 0000-0001-7273-7317, Wing, K, Wang, J-H, Luu, CD, Bender, JA ORCID: 0000-0002-9241-0036, Chen, J, Wang, Q ORCID: 0000-0003-0397-6554, Lu, Q ORCID: 0000-0003-1261-9102, Nguyen Tran, MT, Young, KM ORCID: 0000-0002-1686-3463, Wong, RCB, Pebay, A, Cook, AL ORCID: 0000-0003-1770-7910, Hung, SSC, Liu, G-S ORCID: 0000-0003-3379-724X and Hewitt, AW ORCID: 0000-0002-5123-5999 2020 , 'Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing' , Frontiers in Cellular Neuroscience, vol. 14 , pp. 1-9 , doi:

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CRISPR/Cas has opened the prospect of direct gene correction therapy for someinherited retinal diseases. Previous work has demonstrated the utility of adenoassociatedvirus (AAV) mediated delivery to retinal cells in vivo; however, with theexpanding repertoire of CRISPR/Cas endonucleases, it is not clear which of theseare most efficacious for retinal editing in vivo. We sought to compare CRISPR/Casendonuclease activity using both single and dual AAV delivery strategies for gene editingin retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNAwere generated and validated in YFP-expressing HEK293A cell by flow cytometry andthe T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA wasthen packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally intoCMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiencythan SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editingwas found between single and dual CRISPR/SaCas9 vector systems. With a markedreduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to havethe highest knockout efficacy among all investigated endonucleases. We demonstratethat the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the mostefficient gene modification in neurosensory retinal cells in vivo.

Item Type: Article
Authors/Creators: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
Keywords: CRISPR/Cas, CRISPR (clustered regularly interspaced short palindromic repeats), retina, retinal dystrophy, gene editing, AAV (adeno-associated virus)
Journal or Publication Title: Frontiers in Cellular Neuroscience
Publisher: Frontiers Research Foundation
ISSN: 1662-5102
DOI / ID Number:
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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)

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