Centre for Eye Research Australia (CERA) - Research Publications

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Start date: 2020 × End date: 2021 × Author: Wong, Ching-Bong × Type: Journal Article ×

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    Editorial: Somatic Cell Gene Editing for Treating Diseases
    Wong, RC-B ; Huang, J ; Li, D ; Amaral, O (FRONTIERS MEDIA SA, 2021-12-23)
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    Multi-species single-cell transcriptomic analysis of ocular compartment regulons
    Gautam, P ; Hamashima, K ; Chen, Y ; Zeng, Y ; Makovoz, B ; Parikh, BH ; Lee, HY ; Lau, KA ; Su, X ; Wong, RCB ; Chan, W-K ; Li, H ; Blenkinsop, TA ; Loh, Y-H (NATURE PORTFOLIO, 2021-09-28)
    The retina is a widely profiled tissue in multiple species by single-cell RNA sequencing studies. However, integrative research of the retina across species is lacking. Here, we construct the first single-cell atlas of the human and porcine ocular compartments and study inter-species differences in the retina. In addition to that, we identify putative adult stem cells present in the iris tissue. We also create a disease map of genes involved in eye disorders across compartments of the eye. Furthermore, we probe the regulons of different cell populations, which include transcription factors and receptor-ligand interactions and reveal unique directional signalling between ocular cell types. In addition, we study conservation of regulons across vertebrates and zebrafish to identify common core factors. Here, we show perturbation of KLF7 gene expression during retinal ganglion cells differentiation and conclude that it plays a significant role in the maturation of retinal ganglion cells.
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    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.