Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases.
Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases.
复制标题
DOI：
10.1016/b978-0-12-801185-0.00009-x
复制
发表时间：
2014
影响因子：
--
通讯作者：
Krawczyk, Przemek M.
中科院分区：
文献类型：
--
作者：
Vriend, Lianne E. M.;Jasin, Maria;Krawczyk, Przemek M.
哺乳动物细胞中每天发生数以千计的DNA断裂，包括潜在的致癌双链断裂(DSB)和危险性较小但数量多得多的单链断裂(SSB)。大多数SSB可以很快修复，但也有一些可以转化为DSB，对基因组的完整性构成威胁。虽然SSB通常通过专门的途径修复，但它们也可以触发同源重组(HR)，这是一种通常与DSB修复相关的无错误途径。虽然HR介导的DSB修复已被广泛研究，但HR介导的SSB修复机制尚不清楚。本章描述了一种使用DR-GFP报告程序研究SSB诱导的哺乳动物细胞HR的方法，该报告程序已被广泛用于DSB修复研究，以及一个适应的细菌CRISPR/CAS系统。
Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the integrity of the genome. Although SSBs are usually repaired by dedicated pathways, they can also trigger homologous recombination (HR), an error-free pathway generally associated with DSB repair. While HR-mediated DSB repair has been extensively studied, the mechanisms of HR-mediated SSB repair are less clear. This chapter describes a protocol to investigate SSB-induced HR in mammalian cells employing the DR-GFP reporter, which has been widely used in DSB repair studies, together with an adapted bacterial CRISPR/Cas system.