Centre for Eye Research Australia (CERA) - Research Publications

Permanent URI for this collection

http://hdl.handle.net/11343/198002

Filters

2020 1
1
Reset filters

Settings
Statistics
Citations
Start date: 2020 × Author: Wong, Ching-Bong × Author: Hewitt, Alex ×

Search Results

Now showing 1 - 1 of 1
  • Item
    Thumbnail Image
    Comparison of CRISPR/Cas Endonucleases forin vivoRetinal Gene Editing
    Li, F ; Wing, K ; Wang, J-H ; Luu, CD ; Bender, JA ; Chen, J ; Wang, Q ; Lu, Q ; Nguyen Tran, MT ; Young, KM ; Wong, RCB ; Pebay, A ; Cook, AL ; Hung, SSC ; Liu, G-S ; Hewitt, AW (FRONTIERS MEDIA SA, 2020-09-10)
    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.