Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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DOI：
10.3791/59780
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发表时间：
2019-09-01
影响因子：
1.2
通讯作者：
Moffat, Jason
中科院分区：
文献类型：
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作者：
Chan, Katherine;Tong, Amy Hin Yan;Moffat, Jason
使用CRISPR-CAS系统的基因组编辑极大地提高了精确编辑各种生物基因组的能力。在哺乳动物细胞的背景下，这项技术代表了一种为功能基因组研究进行全基因组遗传筛选的新方法。针对所有开放阅读框架的引导RNA文库(SgRNA)允许在单个细胞池中轻松产生数千种遗传扰动，这些细胞可以筛选特定的表型，以公正和系统的方式涉及基因功能和细胞过程。CRISPR-CAS筛查为研究人员提供了一种简单、高效和廉价的方法来揭示细胞表型的遗传蓝图。此外，对不同细胞系和不同癌症类型的筛查进行的差异分析可以识别在肿瘤细胞中至关重要的基因，揭示特定抗癌治疗的潜在靶点。在人类细胞中进行全基因组筛选可能会令人望而生畏，因为这涉及处理数千万个细胞，并需要分析大量数据。这些筛选的细节，如细胞系特征、CRISPR文库考虑因素，以及在分析过程中对CRISPR技术的限制和能力的理解，往往被忽视。这里提供了成功执行基于CRISPR-CAS9的全基因组池筛查的详细方案。
Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.