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S2R+ cells

Company: Drosophila Genomics Resource Center
Catalog#: 150
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Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells
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2017-02-05
[Abstract]  A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015). [摘要]  合成的致死相互作用是一种遗传相互作用,其中两种基因之一的破坏单独具有影响,但它们的组合破坏是致命的。有关合成致死相互作用的知识可以帮助阐明网络结构和确定人类疾病如癌症的候选药物靶标。在果蝇中,组合基因破坏已经通过组合多个RNAi试剂而实现。这里我们通过组合CRISPR和RNAi来描述高通量组合基因破坏的协议。这种方法以前导致了高度可重现和保守的合成致死相互作用的识别(Housden等人,2015)。

背景 遗传相互作用的知识,如合成致死性,对于确定基因之间的功能关系可能是无价的。例如,酵母中的大规模遗传相互作用屏幕最近被用于组装全球“细胞功能的接线图”(Costanzo等人,2016)。或者,可以使用特定类型的遗传相互作用,例如合成致死相互作用来鉴定包括癌症在内的疾病的药物靶标(Kaelin,2005)。
 鉴定合成相互作用需要组合破坏两个基因。以前在果蝇细胞培养中实现该方法的方法是同时递送多个dsRNA试剂(例如,Fisher等人,2015)。然而,RNAi试剂具有局限性,包括脱靶效应和不完全靶标敲低,当多个试剂一起递送时,RNAi试剂复合。通过将CRISPR诱变与单个dsRNA处理相结合,可以避免这些问题,从而更简单地解释筛选结果并强化“点击”识别。

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