Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.
Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.
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DOI：
10.1016/b978-0-12-801185-0.00012-x
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发表时间：
2014
影响因子：
--
通讯作者：
Brunet, Erika
中科院分区：
文献类型：
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作者：
Renouf, Benjamin;Piganeau, Marion;Ghezraoui, Hind;Jasin, Maria;Brunet, Erika
在许多肿瘤类型中发现了复发性染色体易位，通常导致具有致癌潜力的融合基因的形成和表达。在相关的内源性基因座产生染色体易位，而不仅仅是异位表达融合基因，为更好地表征驱动肿瘤形成的分子机制开辟了新的可能性。在本章中，我们描述了在人类细胞中创建癌症易位的方法。分别由野生型Cas9或Cas9切口酶产生的DSB或配对切口用于诱导相关基因座处的易位。使用不同的基于PCR的方法，我们还解释了如何量化易位频率和分析断点连接在感兴趣的细胞。此外，易位的PCR检测被用作检测脱靶效应的非常灵敏的方法，其具有普遍实用性。
Recurrent chromosomal translocations are found in numerous of tumor types, often leading to the formation and expression of fusion genes with oncogenic potential. Creating chromosomal translocations at the relevant endogenous loci, rather than just ectopically expressing the fusion genes, opens new possibilities for better characterizing molecular mechanisms driving tumor formation. In this chapter, we describe methods to create cancer translocations in human cells. DSBs or paired nicks generated by either wild-type Cas9 or the Cas9 nickase, respectively, are used to induce translocations at the relevant loci. Using different PCR-based methods, we also explain how to quantify translocation frequency and to analyze breakpoint junctions in the cells of interest. In addition, PCR detection of translocations is used as a very sensitive method to detect off-target effects, which has general utility.