Multiplexed CRISPR/Cas9 Targeting of Genes Implicated in Retinal Regeneration and Degeneration.
Multiplexed CRISPR/Cas9 Targeting of Genes Implicated in Retinal Regeneration and Degeneration.
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DOI：
10.3389/fcell.2018.00088
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发表时间：
2018
影响因子：
5.5
通讯作者：
Mumm JS
中科院分区：
文献类型：
--
作者：
Unal Eroglu A;Mulligan TS;Zhang L;White DT;Sengupta S;Nie C;Lu NY;Qian J;Xu L;Pei W;Burgess SM;Saxena MT;Mumm JS
数以千计的基因与视网膜再生有关，但只有少数几个基因被证明影响Müler glia的再生能力--一种具有尚未开发的治疗潜力的成年视网膜干细胞。同样，在与人类视网膜疾病相关的近300个遗传基因座中，大多数仍未在动物模型中进行测试。为了解决这些问题的大规模性质，我们正在斑马鱼中应用基于CRISPR/Cas9的基因组修改策略来针对与视网膜再生或退化有关的300多个基因。我们的目的是通过应用一种显著提高筛查过程效率的多重基因中断策略来实现大规模反向遗传筛查。为了促进大规模的表型鉴定，我们采用了一种基于自动报告量化的分析方法来识别转基因鱼中的细胞退化和再生缺陷表型。多重基因打靶策略可以解决“大数据”生物信息学和湿实验室实验能力之间的规模不匹配问题，这是限制对不断扩大的多组学数据集所涉及的因素进行全面功能分析的一个严重不足。这份报告详细介绍了迄今为止我们在基于CRISPR/Cas9的多重基因靶向策略方面所取得的进展，并讨论了所应用的方法如何促进我们对视网膜退行性疾病易感基因和控制视网膜Müler胶质细胞再生能力的基因的理解。
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.