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ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes

Author

Listed:
  • Kazuto Yoshimi

    (Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
    Mouse Genomics Resource Laboratory, National Institute of Genetics)

  • Yayoi Kunihiro

    (Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
    Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University)

  • Takehito Kaneko

    (Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University)

  • Hitoshi Nagahora

    (BioDynamics Laboratory Inc.)

  • Birger Voigt

    (Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University)

  • Tomoji Mashimo

    (Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
    Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University)

Abstract

The CRISPR-Cas system is a powerful tool for generating genetically modified animals; however, targeted knock-in (KI) via homologous recombination remains difficult in zygotes. Here we show efficient gene KI in rats by combining CRISPR-Cas with single-stranded oligodeoxynucleotides (ssODNs). First, a 1-kb ssODN co-injected with guide RNA (gRNA) and Cas9 messenger RNA produce GFP-KI at the rat Thy1 locus. Then, two gRNAs with two 80-bp ssODNs direct efficient integration of a 5.5-kb CAG-GFP vector into the Rosa26 locus via ssODN-mediated end joining. This protocol also achieves KI of a 200-kb BAC containing the human SIRPA locus, concomitantly knocking out the rat Sirpa gene. Finally, three gRNAs and two ssODNs replace 58-kb of the rat Cyp2d cluster with a 6.2-kb human CYP2D6 gene. These ssODN-mediated KI protocols can be applied to any target site with any donor vector without the need to construct homology arms, thus simplifying genome engineering in living organisms.

Suggested Citation

  • Kazuto Yoshimi & Yayoi Kunihiro & Takehito Kaneko & Hitoshi Nagahora & Birger Voigt & Tomoji Mashimo, 2016. "ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes," Nature Communications, Nature, vol. 7(1), pages 1-10, April.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms10431
    DOI: 10.1038/ncomms10431
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    Cited by:

    1. Kazuto Yoshimi & Kohei Takeshita & Noriyuki Kodera & Satomi Shibumura & Yuko Yamauchi & Mine Omatsu & Kenichi Umeda & Yayoi Kunihiro & Masaki Yamamoto & Tomoji Mashimo, 2022. "Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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