Calarco John A, Norris Adam D
Department of Cell and Systems Biology, University of Toronto, Toronto, Canada.
Department of Biological Sciences, Southern Methodist University, Dallas, United States.
Bio Protoc. 2018 Mar 5;8(5). doi: 10.21769/BioProtoc.2756.
Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and (Schuldiner ., 2005; Butland ., 2008; Costanzo ., 2010), but similar large-scale interaction studies using targeted reverse-genetic deletions in multi-cellular organisms have not been feasible. We developed a CRISPR/Cas9-based method for deleting genes in and replacing them with a heterologous fluorescent reporter (Norris ., 2015). Recently we took advantage of that system to perform a large-scale, reverse genetic screen using null alleles in animals for the first time, focusing on RNA binding protein genes (Norris ., 2017). This type of approach should be similarly applicable to many other gene classes in . Here we detail the protocols involved in generating a library of double mutants and performing medium-throughput competitive fitness assays to test for genetic interactions resulting in fitness changes.
基因相互作用筛选是一种强大的方法，可用于确定基因的新功能并阐明基因之间的功能联系。此类研究在单细胞生物如酵母中取得了显著成效（Schuldiner等人，2005年；Butland等人，2008年；Costanzo等人，2010年），但在多细胞生物中使用靶向反向遗传缺失进行类似的大规模相互作用研究尚不可行。我们开发了一种基于CRISPR/Cas9的方法，用于在动物中删除基因并用异源荧光报告基因进行替换（Norris等人，2015年）。最近，我们首次利用该系统在动物中使用无效等位基因进行大规模反向遗传筛选，重点关注RNA结合蛋白基因（Norris等人，2017年）。这种方法应同样适用于动物中的许多其他基因类别。在这里，我们详细介绍了生成双突变体文库以及进行中通量竞争适应性测定以测试导致适应性变化的基因相互作用所涉及的方案。
