July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Comparison of CRISPR/Cas endonuclease gene editing efficiency of retinal cells in vivo
Author Affiliations & Notes
  • Fan Li
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen University, Guangzhou, Guangdong, China
  • Kristof Wing
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
  • Jiang-Hui Wang
    Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia
    Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, Victoria, Australia
  • James Bender
    Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia
  • Chi D Luu
    Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia
    Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, Victoria, Australia
  • Jinying Chen
    Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
  • Vivienne Lu
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
  • Qi Wang
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
  • Qinyi Lu
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
  • Peter Tran
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
  • Kaylene Young
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
  • Anna King
    Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia
  • Sandy Hung
    Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia
  • Guei-Sheung Liu
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
    Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, Victoria, Australia
  • Alex W Hewitt
    Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia
    Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia
  • Footnotes
    Commercial Relationships   Fan Li, None; Kristof Wing, None; Jiang-Hui Wang, None; James Bender, None; Chi Luu, None; Jinying Chen, None; Vivienne Lu, None; Qi Wang, None; Qinyi Lu, None; Peter Tran, None; Kaylene Young, None; Anna King, None; Sandy Hung, None; Guei-Sheung Liu, None; Alex Hewitt, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4226. doi:
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    • Get Citation

      Fan Li, Kristof Wing, Jiang-Hui Wang, James Bender, Chi D Luu, Jinying Chen, Vivienne Lu, Qi Wang, Qinyi Lu, Peter Tran, Kaylene Young, Anna King, Sandy Hung, Guei-Sheung Liu, Alex W Hewitt; Comparison of CRISPR/Cas endonuclease gene editing efficiency of retinal cells in vivo. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4226.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has opened the prospect of direct gene correction therapy for some inherited retinal disease. Previous work has demonstrated that adeno-associated virus (AAV) mediated in vivo delivery of retinal cells using Streptococcus pyogenes Cas9 (SpCas9). However, with the expanding repertoire of CRISPR/Cas endonucleases, such as Cas12a, Streptococcus aureus Cas9 (SaCas9) and Campylobacter jejuni (CjCas9), it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare the CRISPR/Cas endonuclease activity in retinal cells of CMV-Cre::Rosa26-YFP transgenic mice.

Methods : 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. For each endonuclease, different sgRNAs targeting YFP were designed and validated by FACS and T7E1 assay in Stable YFP expressing HEK293A cells. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2-based variant, AAV7m8, and injected intravitreally into CMV-Cre::Rosa26-YFP mouse. A total of 150 mice were tested, including dual vector with SpCas9 (n=20), SaCas9 (n=20), Cas12a (n=20), CjCas9 (n=41), single vector SaCas9 (n=20) and mCherry control (n=29) experiment cohort. Five months after injection, viral transduction was identified by retinal cryosection and flat-mount. Gene knockout efficacy was determined by quantifying percentage of YFP knockout among the viral infected retinal cells using FACS. One-way ANOVA was used for statistical analysis.

Results : For in vitro validation, SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 (single and dual vector) and CjCas9 in YFP expressing HEK293A cells (Figure 1). AAV7m8-mediated CRISPR/Cas construct achieved effective transduction into outer retina layer and we found a 21.23% (SD=12.17, n=14) reduction of YFP-positive cells in viral-infected retinal cells with SpCas9, with the highest knockout efficacy among all Cas endonucleases, which is consistent with in vitro validation result. However, variation in knockout efficacy with other Cas endonucleases was observed.

Conclusions : We demonstrate that AAV7m8-mediated delivery of CRISPR/Cas constructs have effective transduction and SpCas9 achieves the most efficient gene modification in retinal cells in vivo.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

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