10.3389/fcell.2018.00088.s004 Arife Unal Eroglu Arife Unal Eroglu Timothy S. Mulligan Timothy S. Mulligan Liyun Zhang Liyun Zhang David T. White David T. White Sumitra Sengupta Sumitra Sengupta Cathy Nie Cathy Nie Noela Y. Lu Noela Y. Lu Jiang Qian Jiang Qian Lisha Xu Lisha Xu Wuhong Pei Wuhong Pei Shawn M. Burgess Shawn M. Burgess Meera T. Saxena Meera T. Saxena Jeff S. Mumm Jeff S. Mumm Image_3_Multiplexed CRISPR/Cas9 Targeting of Genes Implicated in Retinal Regeneration and Degeneration.pdf Frontiers 2018 CRISPR/Cas9 zebrafish retina regeneration retinal degeneration large scale multiplex rhodopsin 2018-08-21 07:50:54 Figure https://frontiersin.figshare.com/articles/figure/Image_3_Multiplexed_CRISPR_Cas9_Targeting_of_Genes_Implicated_in_Retinal_Regeneration_and_Degeneration_pdf/6988940 <p>Thousands of genes have been implicated in retinal regeneration, but only a few have been shown to impact the regenerative capacity of Müller glia—an adult retinal stem cell with untapped therapeutic potential. Similarly, among nearly 300 genetic loci associated with human retinal disease, the majority remain untested in animal models. To address the large-scale nature of these problems, we are applying CRISPR/Cas9-based genome modification strategies in zebrafish to target over 300 genes implicated in retinal regeneration or degeneration. Our intent is to enable large-scale reverse genetic screens by applying a multiplexed gene disruption strategy that markedly increases the efficiency of the screening process. To facilitate large-scale phenotyping, we incorporate an automated reporter quantification-based assay to identify cellular degeneration and regeneration-deficient phenotypes in transgenic fish. Multiplexed gene targeting strategies can address mismatches in scale between “big data” bioinformatics and wet lab experimental capacities, a critical shortfall limiting comprehensive functional analyses of factors implicated in ever-expanding multiomics datasets. This report details the progress we have made to date with a multiplexed CRISPR/Cas9-based gene targeting strategy and discusses how the methodologies applied can further our understanding of the genes that predispose to retinal degenerative disease and which control the regenerative capacity of retinal Müller glia cells.</p>