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An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

  1. Oguz Kanca
  2. Jonathan Zirin
  3. Jorge Garcia-Marques
  4. Shannon Marie Knight
  5. Donghui Yang-Zhou
  6. Gabriel Amador
  7. Hyunglok Chung
  8. Zhongyuan Zuo
  9. Liwen Ma
  10. Yuchun He
  11. Wen-Wen Lin
  12. Ying Fang
  13. Ming Ge
  14. Shinya Yamamoto
  15. Karen L Schulze
  16. Yanhui Hu
  17. Allan C Spradling
  18. Stephanie E Mohr
  19. Norbert Perrimon
  20. Hugo J Bellen  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Harvard Medical School, United States
  3. Janelia Research Campus, Howard Hughes Medical Institute, United States
  4. Howard Hughes Medical Institute, Carnegie Institution for Science, United States
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Cite this article as: eLife 2019;8:e51539 doi: 10.7554/eLife.51539

Abstract

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

Article and author information

Author details

  1. Oguz Kanca

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5438-0879
  2. Jonathan Zirin

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  3. Jorge Garcia-Marques

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    No competing interests declared.
  4. Shannon Marie Knight

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  5. Donghui Yang-Zhou

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  6. Gabriel Amador

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  7. Hyunglok Chung

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  8. Zhongyuan Zuo

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  9. Liwen Ma

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  10. Yuchun He

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  11. Wen-Wen Lin

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  12. Ying Fang

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  13. Ming Ge

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  14. Shinya Yamamoto

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  15. Karen L Schulze

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1368-729X
  16. Yanhui Hu

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  17. Allan C Spradling

    Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5251-1801
  18. Stephanie E Mohr

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9639-7708
  19. Norbert Perrimon

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7542-472X
  20. Hugo J Bellen

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    For correspondence
    hbellen@bcm.edu
    Competing interests
    Hugo J Bellen, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5992-5989

Funding

NIH Office of the Director (R01GM067858)

  • Hugo J Bellen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Publication history

  1. Received: September 9, 2019
  2. Accepted: October 31, 2019
  3. Accepted Manuscript published: November 1, 2019 (version 1)

Copyright

© 2019, Kanca et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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