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Arife Eroglu, Timothy Mulligan, Sumitra Sengupta, Jeff S Mumm; Multiplex CRISPR/Cas9-based Genetic Screen for Retinal Regeneration-deficient Zebrafish Mutants. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4475.
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© ARVO (1962-2015); The Authors (2016-present)
Müller glia (MG) cells function as retinal stem cells in zebrafish. Intriguingly, human MG appears to retain regenerative potential; neurons derived from MG in culture can restore visual function when transplanted into retinal degeneration models. Accordingly, the mechanisms regulating the regenerative potential of MG are of great interest. To gain a comprehensive understanding of the factors and cognate signaling systems controlling retinal regeneration, we apply forward and reverse genetics screening approaches to identify regeneration-deficient mutant zebrafish lines. Here, we discuss the use of a multiplexed CRISPR/Cas9-based targeted mutational strategy to evaluate ~300 transcriptomics-implicated genes as factors necessary for rod photoreceptor regeneration in zebrafish.
We have developed complementary cell-specific ablation and whole-organism high-throughput screening (HTS) methods to 1) facilitate studies of cellular regeneration, 2) model degenerative disease, and 3) discover regeneration-promoting factors in zebrafish. Here, a model of retinitis pigmentosa—where apoptosis can be induced specifically in rod cells and a fluorescent reporter allows quantification of cell loss and replacement kinetics—is being used to identify genes required for rod cell regeneration. To implicate genes in rod cell replacement, we performed a microarray analysis comparing two cell-specific ablation paradigms, rod cells versus retinal bipolar cells. Nearly 300 genes upregulated in the rod cell were selected for multiplexed CRISPR/Cas9-based mutational analysis. Using this method, phenotyping assays can be performed in the F1 generation, vastly increasing screening efficiency, and thereby enabling large-scale reverse genetics.
Targeting oligos for 144 candidate genes have been generated. Multiplexing is used to concurrently target 3 gene candidates, resulting in a total of 96 pooled founder groups for 288 gene candidates. Our recent progress in generating mutant lines and identifying regeneration-deficient phenotypes will be discussed.
Multiplexed CRISPR/Cas9 methods enable large-scale mutational analyses, providing a means of functionally evaluating candidate genes at a rate of high-throughput bioinformatics-based datasets. The approaches outlined here can therefore provide fundamental insights into the mechanisms regulating regenerative potential in the retina
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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