From: AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo

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From: AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo Invest. Ophthalmol. Vis. Sci.. 2016;57(7):3470-3476. doi:10.1167/iovs.16-19316 Figure Legend: CRISPR/Cas-mediated gene editing of retinal cells in vivo. Dual-viral suspension of AAV2-SpCas9 and AAV2-sgRNA was used (A). sgRNA plasmids also expressed mCherry, and the size of the cassettes packaged by AAV2 is displayed. Representative retinal montages from Thy1-yellow fluorescent protein (YFP) mice exposed in vivo to our dual AAV2 plasmid system carrying SpCas9 and either control (LacZ) sgRNA (B) or sgRNAs targeting YFP (C) (scale bar: 500 μm). Overall, the proportion of mCherry-expressing cells (mCherry+), which lacked YFP (YFP−), was higher in SpCas9/YFP-sgRNA–treated eyes (D). Higher magnification of flat-mount images, displaying differences in YFP expression following AAV2-mediated delivery of SpCas9/LacZ-sgRNA (E) or SpCas9/YFP-sgRNA (G) (scale bar: 10 μm). Cross sections displaying AAV2 infection of the inner retina confirm YFP knockout in SpCas9/YFP-sgRNA–treated eyes (H), compared to SpCas9/LacZ-sgRNA–treated eyes (F) (scale bar: 50 μm). White arrows indicate AAV2-sgRNA–infected cells. Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ITR; inverted terminal repeat; pMecp2, truncated methyl-CpG-binding protein 2 promoter; HA, hemagglutinin tag; NLS, nuclear localization signal; spA, synthetic polyadenylation signal; U6, Pol III promoter; sgRNA, single guide RNA; hSyn1, human synapsin 1 promoter; mCherry, monomeric cherry fluorescent protein; KASH, Klarsicht ANC1 Syne homology nuclear transmembrane domain; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; bGHpA, bovine growth hormone polyadenylation signal. Date of download: 12/30/2017 The Association for Research in Vision and Ophthalmology Copyright © 2017. All rights reserved.

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