title: Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations.
abstract: 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.
authors.affiliation: Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea.
authors.forename: Suresh
authors.initials: S
authors.lastname: Ramakrishna
authors.affiliation: National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
authors.forename: Seung Woo
authors.initials: SW
authors.lastname: Cho
authors.affiliation: National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
authors.forename: Sojung
authors.initials: S
authors.lastname: Kim
authors.affiliation: Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea.
authors.forename: Myungjae
authors.initials: M
authors.lastname: Song
authors.affiliation: Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea.
authors.forename: Ramu
authors.initials: R
authors.lastname: Gopalappa
authors.affiliation: National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea.
authors.forename: Hyongbum
authors.initials: H
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D058727:RNA, Small Untranslated; D000097573:Red Fluorescent Protein
chemical_list: D064130:CRISPR-Associated Proteins; D004720:Endonucleases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D058727:RNA, Small Untranslated; D000097573:Red Fluorescent Protein