Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.
Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.
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DOI：
10.1016/bs.mcb.2015.03.017
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发表时间：
2015
影响因子：
--
通讯作者：
Holland AJ
中科院分区：
文献类型：
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作者：
Moyer TC;Holland AJ
快速和特异性地修改哺乳动物细胞基因组的能力一直是生物医学研究人员的长期目标。最近，来自细菌的聚集的、有规则间隔的短回文重复序列(CRISPR)/Cas9系统已被用于人类细胞的基因组工程。CRISPR系统将rna引导的Cas9核酸酶引导到特定的基因组位点，诱导DNA双链断裂，随后使用外源DNA修复模板通过同源定向修复进行修复。在这里，我们描述了一种使用CRISPR/Cas9在人类细胞中实现点突变的双等位基因插入的方案。使用该方法，纯合子克隆细胞系可在5-6周内构建。这种方法也适用于在确定的基因组位置插入较大的DNA元素，如荧光蛋白和degrons。CRISPR/Cas9基因组工程在基础科学和转化研究中都提供了令人兴奋的应用。
The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5–6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research.