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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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在人类细胞中使用CRISPR-Cas9系统进行基因选
Tim Wang1, Jenny J Wei, David M Sabatini
1Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
Science (New York, N.Y.)
|December 17, 2013
概括
这项研究引入了哺乳动物遗传学的CRISPR-Cas9选方法,使得有效的基因淘汰和功能分析成为可能. 该方法使用大型单导向RNA库进行聚合基因选,识别DNA修复和细胞循环途径中的关键基因.
科学领域:
- 遗传学 是一个遗传学.
- 分子生物学分子生物学
- 基因组编辑 基因组编辑
背景情况:
- 集群定期间隔的简短的Palindromic重复 (CRISPR) -Cas9系统已经彻底改变了哺乳动物的遗传学.
- 现有的工具允许快速生成转基因细胞系和动物.
研究的目的:
- 开发和验证一个聚合,功能丧失的基因查方法,使用基因组规模的单导向RNA (sgRNA) 库.
- 评估这种选方法在哺乳动物细胞中对阳性和阴性选择的有用性.
主要方法:
- 一个基因组规模的lentiviral sgRNA库,包含73,000个sgRNAs,用于在两个人类细胞系中生成淘汰集.
- 进行了对抗6-thioguanine和etoposide的聚合基因选,以及通过负选择对基本基因进行了基因选.
- 使用大规模并行测序,在稳定整合sgRNA表达盒后跟踪复杂的突变池.
主要成果:
- 对6-thioguanine耐药性的选成功识别了DNA不匹配修复途径的组件.
- 一个检测埃托化物耐药性的镜确定了TOP2A和CDK6.
- 负选择选确定了参与基本细胞过程的基因组,sgRNA效率与特定的序列动机有关.
结论:
- Cas9/sgRNA屏幕代表了哺乳动物细胞遗传分析的强大和系统的方法.
- 开发的选方法对阳性和阴性选择选都有效.
- 根据序列动机预测有效的sgRNA可以提高查效率.
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