Brief Communication | Published:

One-step generation of conditional and reversible gene knockouts

Nature Methods volume 14, pages 287289 (2017) | Download Citation

Abstract

Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR–FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR–Cas9 and a universal conditional intronic cassette.

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Acknowledgements

pPyCAG-eGFP-IRES-Zeo plasmid was kindly provided by A. Smith and pCAGGS-Cre-IRES-Puro and pCAGGS-Flp-IRES-Puro plasmid vectors by B. Hendrich (both at the WT–MRC Cambridge Stem Cell Institute, University of Cambridge). We thank M. Kinoshita for advice regarding antibodies. A.A.-R. and K.T. are supported by the Medical Research Council, A.M. is supported by Wntsapp, Marie Curie ITN. J.F. and J.C.R.S. are supported by the Wellcome Trust. W.C.S. received core grant support from the Wellcome Trust to the Wellcome Trust Sanger Institute. B.-K.K. and R.C.M. are supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (101241/Z/13/Z) and receive a core support grant from the Wellcome Trust and MRC to the WT–MRC Cambridge Stem Cell Institute.

Author information

Author notes

    • Amanda Andersson-Rolf
    • , Roxana C Mustata
    •  & Alessandra Merenda

    These authors contributed equally to this work.

Affiliations

  1. Wellcome Trust–Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.

    • Amanda Andersson-Rolf
    • , Roxana C Mustata
    • , Alessandra Merenda
    • , Jihoon Kim
    • , Katie Tremble
    • , José C R Silva
    • , Juergen Fink
    •  & Bon-Kyoung Koo
  2. Department of Genetics, University of Cambridge, Cambridge, UK.

    • Amanda Andersson-Rolf
    • , Alessandra Merenda
    •  & Bon-Kyoung Koo
  3. Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge, UK.

    • Sajith Perera
    • , Tiago Grego
    • , Katie Andrews
    •  & William C Skarnes
  4. Department of Biochemistry, University of Cambridge, Cambridge, UK.

    • José C R Silva

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Contributions

A.A-R., W.C.S. and B.-K.K. wrote the manuscript. A.A.-R., J.F., W.C.S. and B.-K.K. designed the FLIP cassette targeting vector. A.A.-R., W.C.S. and B.-K.K. designed and discussed the experiments. A.A.-R., R.C.M., and J.K. targeted mESCs and performed WB. A.A.-R. performed immunofluorescence. K.A. and A.M. targeted hiPSCs. A.M. targeted HEK 293 cells. A.A.-R. and A.M. performed FACS. A.A.-R. performed the organoid experiments. S.P. and T.G. performed the bioinformatics analysis. K.T. derived Sox2FLIP/FLIP MEFs. J.C.R.S. supervised K.T. WC.S. and B.-K.K. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to William C Skarnes or Bon-Kyoung Koo.

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DOI

https://doi.org/10.1038/nmeth.4156

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