Suresh Ramakrishna1, Seung Woo Cho2, Sojung Kim2, Myungjae Song1, Ramu Gopalappa1, Jin-Soo Kim2 & Hyongbum Kim1
1 Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea. 2 National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
Correspondence to: Hyongbum Kim or Jin-Soo Kim
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.