title: AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo.
abstract: OBJECTIVE
  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.
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Sandy S C
authors.initials: SS
authors.lastname: Hung
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Vicki
authors.initials: V
authors.lastname: Chrysostomou
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia 2Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia.
authors.forename: Fan
authors.initials: F
authors.lastname: Li
authors.affiliation: Department of Optometry & Vision Sciences, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Jeremiah K H
authors.initials: JK
authors.lastname: Lim
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Jiang-Hui
authors.initials: JH
authors.lastname: Wang
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia 5Centre for Neurogenetics and Statistical Genomics, Queensland Brain Institute, University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Powell
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia 6Department of Ophthalmology, Jinan University, Guangzhou, Guangdong, China.
authors.forename: Leilei
authors.initials: L
authors.lastname: Tu
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Maciej
authors.initials: M
authors.lastname: Daniszewski
authors.affiliation: Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia.
authors.forename: Camden
authors.initials: C
authors.lastname: Lo
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Wong
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Crowston
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Alice
authors.initials: A
authors.lastname: Pébay
authors.affiliation: Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, Tasmania, Australia.
authors.forename: Anna E
authors.initials: AE
authors.lastname: King
authors.affiliation: Department of Optometry & Vision Sciences, University of Melbourne, Melbourne, Victoria, Australia.
authors.forename: Bang V
authors.initials: BV
authors.lastname: Bui
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia.
authors.forename: Guei-Sheung
authors.initials: GS
authors.lastname: Liu
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia 2Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia.
authors.forename: Alex W
authors.initials: AW
authors.lastname: Hewitt
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000229:Dependovirus / Q000235:genetics*; D004596:Electroretinography; D000072669:Gene Editing / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D056965:Injections, Intraocular; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010957:Plasmids; D012160:Retina / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria