Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.
Enriching CRISPR-Cas9 targeted cells by co-targeting the HPRT gene.
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DOI：
10.1093/nar/gkv675
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发表时间：
2015-11-16
影响因子：
14.9
通讯作者：
Yan H
中科院分区：
文献类型：
--
作者：
Liao S;Tammaro M;Yan H
CRISPR-Cas9系统使用向导RNA来指导Cas9内切核酸酶切割靶序列。理论上，它可以靶向基因组中的任何序列，但预测的指导RNA的效率差异很大。如果没有获得靶细胞，也很难知道实验失败的原因。我们已经开发了一种基于瞬时转染的方法，通过共靶向次黄嘌呤磷酸核糖基转移酶（HPRT）基因来富集成功靶向的细胞。用分别靶向HPRT和感兴趣的基因的两种指导RNA转染细胞。通过对6-硫鸟嘌呤（6-TG）的抗性选择HPRT靶向细胞，然后检查共转染的指导RNA靶向的基因的潜在改变。AAVS 1、Exo 1和Trex 1等基因的改变在6-TG耐药细胞中高度富集。该方法在HCT 116细胞和U2 OS细胞中均有效，并且可以容易地按比例放大以加工多种指导RNA。当共靶向失败时，可以直接确定靶基因是必需的还是指导RNA无效。因此，HPRT共靶向提供了一种简单、有效和可扩展的方式来富集基因靶向事件并鉴定失败的原因。
The CRISPR-Cas9 system uses guide RNAs to direct the Cas9 endonuclease to cleave target sequences. It can, in theory, target essentially any sequence in a genome, but the efficiency of the predicted guide RNAs varies dramatically. If no targeted cells are obtained, it is also difficult to know why the experiment fails. We have developed a transient transfection based method to enrich successfully targeted cells by co-targeting the hypoxanthine phosphoribosyltransferase (HPRT) gene. Cells are transfected with two guide RNAs that target respectively HPRT and the gene of interest. HPRT targeted cells are selected by resistance to 6-thioguanine (6-TG) and then examined for potential alterations to the gene targeted by the co-transfected guide RNA. Alterations of many genes, such as AAVS1, Exo1 and Trex1, are highly enriched in the 6-TG resistant cells. This method works in both HCT116 cells and U2OS cells and can easily be scaled up to process multiple guide RNAs. When co-targeting fails, it is straightforward to determine whether the target gene is essential or the guide RNA is ineffective. HPRT co-targeting thus provides a simple, efficient and scalable way to enrich gene targeting events and to identify the cause of failure.