Protocol

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors

  • Nature Protocols volume 13, pages 195215 (2018)
  • doi:10.1038/nprot.2017.153
  • Download Citation
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Abstract

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1–10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts–CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30–60% and reaches as high as 100% in some cases). The protocol takes 2 months to generate the founder mice.

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Acknowledgements

This work was supported in part by a Grant-in-Aid for Young Scientists (B) (16K18821) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to H.M.; and by 2014 Tokai University School of Medicine Research Aid, the MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2015–2019 to Tokai University, the Research and Study Project of Tokai University General Research Organization, a Grant-in-Aid for Scientific Research (16H04685) from the MEXT, and funding from 2016–2017 Tokai University School of Medicine Project Research to M.O.; and by an Institutional Development Award (principal investigator: S. Smith) P20GM103471 (to C.B.G. and R.M.Q.). We thank A. Koesters, University of Nebraska Medical Center, for her editorial contribution and J.M. Miano, University of Rochester, for his helpful comments on the manuscript. We also gratefully acknowledge the contribution of the staff of the Support Center for Medical Research and Education, Tokai University, for sequencing and microinjection.

Author information

Author notes

    • Hiromi Miura
    •  & Rolen M Quadros

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.

    • Hiromi Miura
    •  & Masato Ohtsuka
  2. Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Kanagawa, Japan.

    • Hiromi Miura
    •  & Masato Ohtsuka
  3. Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, Nebraska, USA.

    • Rolen M Quadros
    •  & Channabasavaiah B Gurumurthy
  4. Developmental Neuroscience, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, Nebraska, USA.

    • Channabasavaiah B Gurumurthy
  5. The Institute of Medical Sciences, Tokai University, Kanagawa, Japan.

    • Masato Ohtsuka

Authors

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Contributions

M.O. conceived the idea of using long ssDNAs as donors in genome editing, which was further tested and improved upon by the other three authors. All authors contributed equally in writing the manuscript.

Competing interests

C.B.G., M.O., and H.M. have filed a patent application relating to the work described in this paper with international application number PCT/US2016/035660, filed June 3, 2016 (DNA editing using single-stranded DNA).

Corresponding authors

Correspondence to Channabasavaiah B Gurumurthy or Masato Ohtsuka.

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    Supplementary Text and Figures

    Supplementary Figures 1–5 and Supplementary Table 1.

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