Article

Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations

  • Nature Communications 5, Article number: 3378 (2014)
  • doi:10.1038/ncomms4378
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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.

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Acknowledgements

We are grateful to H.K.'s lab members for their suggestions and discussions on this project. H.K. and J.-S.K. are supported in part by the National Research Foundation (H.K., 2011-0013568, 2008-0062287, 2011-0019357, 2013M3A9B4076544; J.-S.K., 2013-000718) and Converging Research Center Program (H.K., 2013K000275) funded by the Ministry of Science, ICT, and Future Planning, and the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (H.K., H10C1740).

Author information

Affiliations

  1. Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seongdong-gu, Seoul 133-791, South Korea

    • Suresh Ramakrishna
    • , Myungjae Song
    • , Ramu Gopalappa
    •  & Hyongbum Kim
  2. National Creative Research Initiatives Center for Genome Engineering and Department of Chemistry, Seoul National University, Gwanak-gu, Seoul 151-747, South Korea

    • Seung Woo Cho
    • , Sojung Kim
    •  & Jin-Soo Kim

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Contributions

Conceived and designed the experiments: S.R., H.K. and J.-S.K.; performed the experiments: S.R., S.W.C., S.K., S.M. and R.G.; analysed the data: S.R. and H.K.; wrote the paper: H.K., S.R. and J.-S.K.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jin-Soo Kim or Hyongbum Kim.

Supplementary information

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    Supplementary Information

    Supplementary Figures 1-15 and Supplementary Tables 1-5

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