Protocol | Published:

Precise and efficient scarless genome editing in stem cells using CORRECT

Nature Protocols volume 12, pages 329354 (2017) | Download Citation

Abstract

CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated—e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes—for example, in human induced pluripotent (iPS) stem cells. This protocol outlines in detail how to implement either the re-Guide or re-Cas variants of CORRECT to generate scarlessly edited isogenic stem cell lines with intended monoallelic and biallelic sequence changes in 3 months.

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Acknowledgements

This research was supported by The Rockefeller University, The New York Stem Cell Foundation, The Ellison Foundation, the Cure Alzheimer's Fund and the Empire State Stem Cell fund through New York State Department of Health. D.K. is a Howard Hughes Medical Institute International Student Research Fellow and received a fellowship from the National Sciences and Engineering Research Council of Canada. D.P. is a New York Stem Cell Foundation Druckenmiller Fellow and received a fellowship from the German Academy of Sciences Leopoldina. S.T. is supported by the Agency for Science, Technology and Research of Singapore. We thank A. Sproul, S. Jacob and S. Noggle for sharing protocols and know-how for stem cell culture and for sharing stem cell lines. We thank members of the Tessier-Lavigne laboratory for discussions. Our thanks also go to S. Mazel and the team at the Rockefeller University Flow Cytometry Resource Center, C. Zhao and the team at the Rockefeller University Genomics Resource Center, and A. Chen and M. Duffield for technical help. Opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Fund, the New York State Department of Health or the State of New York.

Author information

Author notes

    • Dylan Kwart
    •  & Dominik Paquet

    These authors contributed equally to this work.

Affiliations

  1. Laboratory for Brain Development and Repair, The Rockefeller University, New York, USA.

    • Dylan Kwart
    • , Dominik Paquet
    • , Shaun Teo
    •  & Marc Tessier-Lavigne

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Contributions

D.K., D.P. and M.T.-L. conceived and designed the protocol. D.K. and D.P. performed and analyzed the experiments. S.T. helped analyze NGS data. D.K., D.P. and M.T.-L. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Marc Tessier-Lavigne.

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    sgRNA, primer and ssODN repair template sequences used in our experiments.

About this article

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DOI

https://doi.org/10.1038/nprot.2016.171

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