Data_Sheet_1_Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing.docx (2.23 MB)
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Data_Sheet_1_Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing.docx

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posted on 2020-09-10, 04:48 authored by Fan Li, 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 C. B. Wong, Alice Pébay, Anthony L. Cook, Sandy S. C. Hung, Guei-Sheung Liu, Alex W. Hewitt

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.