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

By Guei-Sheung Liu, Kristof Wing, Jiang-Hui Wang, Chi D Luu, James A Bender, Jinying Chen, Qi Wang, Qinyi Lu, Minh Thuan Nguyen Tran, Kaylene M Young, Raymond Ching-Bong Wong, Alice Pebay, Anthony L Cook, Sandy SC Hung, Guei-Sheung Liu, Alex Hewitt

Posted 09 Jun 2020
bioRxiv DOI: 10.1101/2020.06.09.141705 (published DOI: 10.3389/fncel.2020.570917)

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. ### Competing Interest Statement The authors have declared no competing interest.

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