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Genome Biol. 2018 Feb 26;19(1):25. doi: 10.1186/s13059-018-1400-x.

i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases.

Author information

1
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Isehara, Kanagawa, Japan. masato@is.icc.u-tokai.ac.jp.
2
Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, Japan. masato@is.icc.u-tokai.ac.jp.
3
The Institute of Medical Sciences, Tokai University, Isehara, Kanagawa, Japan. masato@is.icc.u-tokai.ac.jp.
4
Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
5
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Isehara, Kanagawa, Japan.
6
Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, Japan.
7
Laboratory Animal Facilities & Services, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
8
Division of Molecular Genetics, Shigei Medical Research Institute, Minami-ku, Okayama, Japan.
9
Department of Applied Biochemistry, School of Engineering, Tokai University, Hiratsuka, Kanagawa, Japan.
10
Division of Biomedical Engineering, National Defense Medical College Research Institute, Tokorozawa, Saitama, Japan.
11
Department of Bioproduction, Tokyo University of Agriculture, Abashiri, Hokkaido, Japan.
12
Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
13
Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE, USA.

Abstract

We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.

KEYWORDS:

CRISPR; Easi-CRISPR; GONAD; In vivo electroporation; Knock-in; Long ssDNA; Transgenic mouse

PMID:
29482575
PMCID:
PMC5828090
DOI:
10.1186/s13059-018-1400-x
[Indexed for MEDLINE]
Free PMC Article

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