Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines
Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines
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DOI：
10.1016/j.ymeth.2017.05.003
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发表时间：
2017-05-15
期刊：
影响因子：
4.8
通讯作者：
Margueron, R.
中科院分区：
文献类型：
--
作者：
Wassef, M.;Luscan, A.;Margueron, R.
ZFN、TALEN和CRISPR/Cas9等可编程核酸酶的出现为广泛使用的模型系统带来了遗传操纵的力量。在哺乳动物细胞中，核酸酶介导的DNA双链断裂主要通过易错非同源末端连接（NHEJ）修复途径修复，最终导致可破坏基因功能的小缺失或插入（indels）的积累。然而，由于插入缺失的可变大小和许多细胞系的多倍体状态（例如，癌症来源的细胞），获得敲除通常需要冗长的筛选和表征程序。考虑到同源定向修复（HDR）可以引入更精确的修饰类型，我们开发了基于HDR的基因靶向策略，大大促进了细胞系中敲除生成的过程。为了产生可逆敲除（R-KO），将可选择的无启动子的STOP盒插入内含子中，中断转录。功能丧失可以通过RT-qPCR进行验证，并且是可去除的，从而能够随后恢复基因功能。R-KO程序的变体可用于引入点突变。为了产生组成型敲除（C-KO），靶向外显子，其利用基于HDR的基因破坏以及非HDR靶向等位基因上的NHEJ诱导的插入缺失。因此，C-KO程序极大地促进了多个等位基因的同时失活。总的来说，这些基因组编辑工具为功能遗传学方法提供了上级精度和效率。我们提供了详细的协议指导靶向载体的设计和基因靶向实验的分析和验证。(C)2017爱思唯尔公司All rights reserved.
The advent of programmable nucleases such as ZFNs, TALENs and CRISPR/Cas9 has brought the power of genetic manipulation to widely used model systems. In mammalian cells, nuclease-mediated DNA double strand break is mainly repaired through the error-prone non-homologous end joining (NHEJ) repair pathway, eventually leading to accumulation of small deletions or insertions (indels) that can inactivate gene function. However, due to the variable size of the indels and the polyploid status of many cell lines (e.g., cancer-derived cells), obtaining a knockout usually requires lengthy screening and characterization procedures. Given the more precise type of modifications that can be introduced upon homology-directed repair (HDR), we have developed HDR-based gene-targeting strategies that greatly facilitate the process of knockout generation in cell lines. To generate reversible knockouts (R-KO), a selectable promoter-less STOP cassette is inserted in an intron, interrupting transcription. Loss-of-function can be validated by RT-qPCR and is removable, enabling subsequent restoration of gene function. A variant of the R-KO procedure can be used to introduce point mutations. To generate constitutive knockouts (C-KO), an exon is targeted, which makes use of HDR-based gene disruption together with NHEJ-induced indels on nonHDR targeted allele(s). Hence the C-KO procedure greatly facilitates simultaneous inactivation of multiple alleles. Overall these genome-editing tools offer superior precision and efficiency for functional genetic approaches. We provide detailed protocols guiding in the design of targeting vectors and in the analysis and validation of gene targeting experiments. (C) 2017 Elsevier Inc. All rights reserved.