An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein
An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein
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DOI：
10.7554/elife.35069
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发表时间：
2018-04-11
期刊：
影响因子：
7.7
通讯作者：
Pollard, Steven M.
中科院分区：
文献类型：
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作者：
Dewari, Pooran Singh;Southgate, Benjamin;Pollard, Steven M.
CRISPR/Cas9可用于将表位标签精确地遗传敲入内源基因，简化蛋白质功能的实验分析。然而，在原代哺乳动物细胞培养中，Cas9辅助的表位标记通常是低效的，并且依赖于基于质粒的选择策略。在这里，我们证明了提高不同标签(V5，3XFLAG，Myc，HA)的敲入效率，方法是将Cas9蛋白与两部分合成的修饰RNA(退火的crRNA：trrRNA)和单链寡核苷酸(SsODN)修复模板共同传递。在胚胎干细胞(ES)、神经干细胞(NS)和脑肿瘤来源的干细胞中，未经选择的情况下，转基因效率达到了5%-30%。双等位基因标记的克隆系很容易获得，并用来定义寡核苷酸结合染色质的相互作用伙伴。使用我们新颖的基于网络的设计工具，我们建立了一个96孔格式的管道，可以对60种不同的转录因子进行V5标记。这种高效、免选择和可扩展的表位标记管道使系统地研究不同哺乳动物干细胞的蛋白质表达水平、亚细胞定位和相互作用成为可能。
CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of 5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.