Sandy S C Hung1, Vicki Chrysostomou1, Fan Li2, Jeremiah K H Lim3, Jiang-Hui Wang1, Joseph E Powell4, Leilei Tu5, Maciej Daniszewski1, Camden Lo6, Raymond C Wong1, Jonathan G Crowston1, Alice Pébay1, Anna E King7, Bang V Bui3, Guei-Sheung Liu1, Alex W Hewitt2. 1. Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia. 2. 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. 3. Department of Optometry & Vision Sciences, University of Melbourne, Melbourne, Victoria, Australia. 4. 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. 5. 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. 6. Monash Micro Imaging, Monash University, Melbourne, Victoria, Australia. 7. Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, Tasmania, Australia.
Abstract
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.
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 transgenicmouse, 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 transgenicmouse 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: Thiago Cabral; James E DiCarlo; Sally Justus; Jesse D Sengillo; Yu Xu; Stephen H Tsang Journal: Curr Opin Ophthalmol Date: 2017-05 Impact factor: 3.761
Authors: Pingjuan Li; Benjamin P Kleinstiver; Mihoko Y Leon; Michelle S Prew; Daniel Navarro-Gomez; Scott H Greenwald; Eric A Pierce; J Keith Joung; Qin Liu Journal: CRISPR J Date: 2018-02
Authors: Erin R Burnight; Joseph C Giacalone; Jessica A Cooke; Jessica R Thompson; Laura R Bohrer; Kathleen R Chirco; Arlene V Drack; John H Fingert; Kristan S Worthington; Luke A Wiley; Robert F Mullins; Edwin M Stone; Budd A Tucker Journal: Prog Retin Eye Res Date: 2018-03-22 Impact factor: 21.198
Authors: K Tyler McCullough; Sanford L Boye; Diego Fajardo; Kaitlyn Calabro; James J Peterson; Christianne E Strang; Dibyendu Chakraborty; Sebastian Gloskowski; Scott Haskett; Steven Samuelsson; Haiyan Jiang; C Douglas Witherspoon; Paul D Gamlin; Morgan L Maeder; Shannon E Boye Journal: Hum Gene Ther Date: 2018-12-20 Impact factor: 5.695