Multiplexed CRISPR/Cas9 Targeting of Genes Implicated in Retinal Regeneration and Degeneration

Abstract

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.

Large-scale Phenotypic Drug Screen Identifies Neuroprotectants in Zebrafish and Mouse Models of Retinitis Pigmentosa

“…Major aspects of retinal cytogenesis are largely complete by 5 days post-fertilization (dpf) in zebrafish (Schmitt and Dowling, 1999;Stenkamp, 2011). Reporter expression in rho:YFP-NTR larvae has also stabilized by this time point (Unal Eroglu et al, 2018), consistent with rho expression (Raymond et al, 1995). We therefore chose 5 dpf to initiate Mtz-induced rod cell ablation.…”

“…Zebrafish Tg(rho:YFP-NTR) larvae were treated with 2.5 mM Mtz or 0.1% DMSO at 5 dpf for 2 days, and collected at 7 dpf for immunostaining using previously reported methods (Unal Eroglu et al, 2018). Briefly, larvae were fixed in 4% PFA at 4˚C overnight, infiltrated with 30% sucrose, and embedded in OCT. Cryosections of 10 µm thickness were made for immunofluorescence staining.…”

Model systems for regeneration: zebrafish

“…A better approach to understanding the effect of the downregulation of a specific gene relies on the use of precise targeted gene editing tools such as TALENs and CRISPR/Cas9. These have proven useful for generating null mutants in zebrafish (Hwang et al, 2013;Jao et al, 2013) and for assessing gene function during regeneration (Dupret et al, 2017;Dong et al, 2019;Nauroy et al, 2019;Unal Eroglu et al, 2018). The generation of conditional knockouts has also recently been achieved by the directed insertion of loxP sites using CRISPR/Cas9 (Burg et al, 2018).…”

Zebrafish Photoreceptor Degeneration and Regeneration Research to Understand Hereditary Human Blindness

“…A screening method for hair cell regeneration identified 7 genes involved in this response [100]. To evaluate genes important for retinal regeneration, large-scale, reverse genetic screening has been established by applying a multiplexed gene disruption strategy [101]. This screening used an automated reporter quantification-based assay to identify cellular regeneration-deficient phenotypes in transgenic fish.…”