Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps.
Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps.
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DOI：
10.1038/nprot.2014.103
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发表时间：
2014-08
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
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作者：
微生物系统遗传互作图谱是鉴定基因间功能关系和确定未鉴定基因功能的有力工具。我们最近在哺乳动物细胞中实施了这一策略，作为一个两阶段的方法。首先，使用复杂的shRNA文库在合并的全基因组筛选中稳健地鉴定感兴趣的基因。其次，在双shRNA筛选中测量命中基因的所有成对组合的表型，并用于构建遗传相互作用图谱。我们的协议允许在各种条件下进行快速汇总筛选，而不需要机器人，与阵列方法相反。该方案的每个阶段可在约2周内实施，并有额外的时间用于分析和生成试剂。我们讨论了屏幕设计的考虑因素，并提出了完整的实验程序，以及一个完整的计算分析套件，用于识别在合并屏幕和生成的遗传相互作用图的命中。虽然这里概述的方案是为我们最初的基于shRNA的方法开发的，但它们可以更广泛地应用，包括基于CRISPR的方法。
Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of hit genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each stage of the protocol can be implemented in ~2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and present complete experimental procedures as well as a full computational analysis suite for identification of hits in pooled screens and generation of genetic interaction maps. While the protocols outlined here were developed for our original shRNA-based approach, they can be applied more generally, including to CRISPR-based approaches.