创建RAW264.7 CRISPR-Cas9全基因组文库。
Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library.
作者信息
Napier Brooke A, Monack Denise M
机构信息
Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA.
出版信息
Bio Protoc. 2017 May 20;7(10). doi: 10.21769/BioProtoc.2320.
The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier ., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.
细菌的成簇规律间隔短回文重复序列（CRISPR）-Cas9基因组编辑工具用于哺乳动物细胞，以敲除特定的目标基因来阐明基因功能。CRISPR-Cas9系统要求哺乳动物细胞表达Cas9核酸内切酶、引导核酸内切酶作用于目标基因的引导RNA（gRNA）以及将Cas9与gRNA连接起来的PAM序列。CRISPR-Cas9全基因组文库用于以无偏倚的高通量方式筛选基因组中每个基因对感兴趣的细胞表型的影响。在本方案中，我们描述了在转化的小鼠巨噬细胞系（RAW264.7）中创建CRISPR-Cas9全基因组文库的方法。我们已利用该文库鉴定了半胱天冬酶-11细胞死亡途径中的新型介质（内皮尔，2016年）；然而，该文库随后可用于筛选多个小鼠巨噬细胞细胞途径中特定基因的重要性。
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