Genome Engineering Using CRISPR-Cas9 System
Genome Engineering Using CRISPR-Cas9 System
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DOI：
10.1007/978-1-4939-1862-1_10
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发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Zhang, Feng
中科院分区：
文献类型：
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作者：
Cong, Le;Zhang, Feng
CRISPR -Cas9系统是一种存在于多种微生物中的适应性免疫系统。它可以在真核细胞中作为一种快速、低成本、高效和可扩展的工具来操作基因组序列。在本章中，详细描述了利用化脓性链球菌的CRISPR-Cas9系统在哺乳动物细胞中实现rna引导的基因组工程应用的方案。我们介绍了所有相关的方法，包括初始位点选择、分子克隆、引导rna （gRNAs）和Cas9进入哺乳动物细胞、靶切割验证以及检测基因组修饰（包括indel和同源重组）的分析。这些工具为研究人员提供了加速正向和反向遗传学研究的新工具。
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.