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140830 - Comparison of CRISPR.pdf (3.65 MB)

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

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journal contribution
posted on 2023-05-20, 17:39 authored by Li, F, Kristof WingKristof Wing, Wang, J-H, Luu, CD, James BenderJames Bender, Chen, J, Wang, Q, Lu, Q, Minh Thuan Nguyen Tran, Kaylene YoungKaylene Young, Wong, RCB, Pebay, A, Anthony CookAnthony Cook, Hung, SSC, Guei-Sheung LiuGuei-Sheung Liu, Alexander HewittAlexander Hewitt
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.

Funding

National Health & Medical Research Council

History

Publication title

Frontiers in Cellular Neuroscience

Volume

14

Article number

570917

Number

570917

Pagination

1-9

ISSN

1662-5102

Department/School

Menzies Institute for Medical Research

Publisher

Frontiers Research Foundation

Place of publication

Switzerland

Rights 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/

Repository Status

  • Open

Socio-economic Objectives

Clinical health not elsewhere classified; Expanding knowledge in the environmental sciences

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