通过比较单倍体和 CRISPR 正向遗传筛选对哺乳动物 ERAD 的遗传剖析。
Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens.
机构信息
Department of Medicine, Cambridge Institute for Medical Research, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY, UK.
Department of Biology, University of Copenhagen, 2200 Copenhagen N, Denmark.
出版信息
Nat Commun. 2016 Jun 10;7:11786. doi: 10.1038/ncomms11786.
The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing 'gold standard' method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.
正向遗传筛选在培养的人类细胞中的应用代表了研究基因功能的一种强大方法。细菌 CRISPR/Cas9 系统的重新利用为在哺乳动物细胞中破坏基因功能提供了一种有效方法，并已应用于全基因组筛选。在这里，我们比较了全基因组 CRISPR/Cas9 介导的正向遗传筛选与基因陷阱诱变筛选在单倍体人类细胞中的功效，单倍体细胞筛选代表了现有的“黄金标准”方法。这种直接比较旨在鉴定 MHC I 分子内质网相关降解（ERAD）所需的基因。这两种方法具有高度一致性（>70%），成功鉴定了经典糖蛋白 ERAD 途径的大多数已知成分。两种筛选方法都鉴定了一个未被表征的基因 TXNDC11 的作用，我们发现它编码一种 EDEM2/3 相关的二硫键还原酶。全基因组 CRISPR/Cas9 介导的筛选与单倍体遗传筛选一起为正向遗传工具包提供了强大的补充。
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