Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea.
National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
Nat Commun. 2014 Feb 26;5:3378. doi: 10.1038/ncomms4378.
RNA-guided endonucleases (RGENs), which are based on the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system, have recently emerged as a simple and efficient tool for genome editing. However, the activities of prepared RGENs are sometimes low, hampering the generation of cells containing RGEN-induced mutations. Here we report efficient methods to enrich cells containing RGEN-induced mutations by using surrogate reporters. HEK293T cells are cotransfected with the reporter plasmid, a plasmid encoding Cas9 and a plasmid encoding crRNA and tracrRNA, and subjected to flow cytometric sorting, magnetic separation or hygromycin selection. The selected cell populations are highly enriched with cells containing RGEN-induced mutations, by a factor of up to 11-fold as compared with the unselected population. The fold enrichment tends to be high when RGEN activity is low. We envision that these reporters will facilitate the use of RGEN in a wide range of biomedical research.
RNA 引导的内切酶（RGENs）是基于成簇、规律间隔的短回文重复序列（CRISPR）-CRISPR 相关（Cas）系统的，最近已成为基因组编辑的一种简单而高效的工具。然而，制备的 RGEN 的活性有时较低，这阻碍了含有 RGEN 诱导突变的细胞的产生。在这里，我们报告了通过使用替代报告基因来富集含有 RGEN 诱导突变的细胞的有效方法。将报告基因质粒、编码 Cas9 的质粒和编码 crRNA 和 tracrRNA 的质粒共转染 HEK293T 细胞，并进行流式细胞术分选、磁分离或潮霉素选择。与未选择的细胞群体相比，通过高达 11 倍的倍数富集选择细胞群体中含有 RGEN 诱导突变的细胞。当 RGEN 活性较低时，富集倍数往往较高。我们设想这些报告基因将促进 RGEN 在广泛的生物医学研究中的应用。
