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AAV-mediated CRISPR/Cas gene editing of retinal cells in vivo


Hung, SSC and Chrysostomou, V and Li, F and Lim, JKH and Wang, J-H and Powell, JE and Tu, L and Daniszewski, M and Lo, C and Wong, RC and Crowston, JG and Pebay, A and King, AE and Bui, BV and Liu, G-S and Hewitt, AW, AAV-mediated CRISPR/Cas gene editing of retinal cells in vivo, Investigative Ophthalmology & Visual Science, 57, (7) pp. 3470-3476. ISSN 1552-5783 (2016) [Refereed Article]


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Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

DOI: doi:10.1167/iovs.16-19316


Purpose: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo.

Methods: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts.

Results: Adeno-associated virus 2–mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8–86.9) reduction of YFP-positive cells in YFP-sgRNA–infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes.

Conclusions: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

Item Details

Item Type:Refereed Article
Keywords:gene editing, CRISPR, Cas9, in vivo, retina
Research Division:Biomedical and Clinical Sciences
Research Group:Neurosciences
Research Field:Cellular nervous system
Objective Division:Health
Objective Group:Clinical health
Objective Field:Clinical health not elsewhere classified
UTAS Author:Li, F (Dr Fan Li)
UTAS Author:King, AE (Professor Anna King)
UTAS Author:Liu, G-S (Associate Professor Guei-Sheung Liu)
UTAS Author:Hewitt, AW (Professor Alex Hewitt)
ID Code:109975
Year Published:2016
Web of Science® Times Cited:86
Deposited By:Wicking Dementia Research and Education Centre
Deposited On:2016-07-09
Last Modified:2022-08-25
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