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

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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.

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Most cited references 14

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

Hiromi Miura, Rolen M Quadros, Channabasavaiah Gurumurthy … (2017)

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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Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease

Wenning Qin, Stephanie Dion, Peter Kutny … (2015)

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

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Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.

Masakazu Hashimoto, Yukiko Yamashita, Tatsuya Takemoto (2016)

The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.

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Author and article information

Contributors

Masato Ohtsuka:

ORCID: http://orcid.org/0000-0002-6952-4238

masato@is.icc.u-tokai.ac.jp

Journal

Journal ID (nlm-ta): Genome Biol

Journal ID (iso-abbrev): Genome Biol

Title: Genome Biology

Publisher: BioMed Central (London )

ISSN (Print): 1474-7596

ISSN (Electronic): 1474-760X

Publication date (Electronic): 26 February 2018

Publication date PMC-release: 26 February 2018

Publication date Collection: 2018

Volume: 19

Electronic Location Identifier: 25

Affiliations

[1 ]ISNI 0000 0001 1516 6626, GRID grid.265061.6, Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, , School of Medicine, Tokai University, ; Isehara, Kanagawa Japan

[2 ]ISNI 0000 0001 1516 6626, GRID grid.265061.6, Center for Matrix Biology and Medicine, Graduate School of Medicine, , Tokai University, ; Isehara, Kanagawa Japan

[3 ]ISNI 0000 0001 1516 6626, GRID grid.265061.6, The Institute of Medical Sciences, , Tokai University, ; Isehara, Kanagawa Japan

[4 ]ISNI 0000 0001 1167 1801, GRID grid.258333.c, Section of Gene Expression Regulation, , Frontier Science Research Center, Kagoshima University, ; Kagoshima, Japan

[5 ]ISNI 0000 0004 1762 0759, GRID grid.411951.9, Laboratory Animal Facilities & Services, Preeminent Medical Photonics Education & Research Center, , Hamamatsu University School of Medicine, ; Hamamatsu, Shizuoka, Japan

[6 ]ISNI 0000 0004 0377 284X, GRID grid.415729.c, Division of Molecular Genetics, Shigei Medical Research Institute, ; Minami-ku, Okayama, Japan

[7 ]ISNI 0000 0001 1516 6626, GRID grid.265061.6, Department of Applied Biochemistry, School of Engineering, , Tokai University, ; Hiratsuka, Kanagawa Japan

[8 ]ISNI 0000 0004 0374 0880, GRID grid.416614.0, Division of Biomedical Engineering, , National Defense Medical College Research Institute, ; Tokorozawa, Saitama, Japan

[9 ]GRID grid.410772.7, Department of Bioproduction, , Tokyo University of Agriculture, ; Abashiri, Hokkaido Japan

[10 ]ISNI 0000 0001 0666 4105, GRID grid.266813.8, Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, , University of Nebraska Medical Center, ; Omaha, NE USA

[11 ]ISNI 0000 0001 0666 4105, GRID grid.266813.8, Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, , University of Nebraska Medical Center, ; Omaha, NE USA

Author information

Masato Ohtsuka http://orcid.org/0000-0002-6952-4238

Article

Publisher ID: 1400

DOI: 10.1186/s13059-018-1400-x

PMC ID: 5828090

PubMed ID: 29482575

SO-VID: 24733204-a9a9-45fe-973e-57a68d3c0679

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 26 September 2017

Date accepted : 30 January 2018

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;

Award ID: 16K18821

Award ID: 15K14371

Award ID: 16H05049

Award Recipient : Masato Ohtsuka Hiromi Miura Shingo Nakamura

Funded by: Tokai University School of Medicine Research Aid

Award ID: 2014

Award Recipient : Masato Ohtsuka

Funded by: Tokai University School of Medicine Project Research

Award ID: 2016-2017

Award Recipient : Masato Ohtsuka

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ScienceOpen disciplines: Genetics

Keywords: in vivo electroporation,crispr,gonad,knock-in,transgenic mouse,long ssdna,easi-crispr

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ScienceOpen disciplines: Genetics

Keywords: in vivo electroporation, crispr, gonad, knock-in, transgenic mouse, long ssdna, easi-crispr

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

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Genome Engineering using CRISPR

Most cited references 14

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

Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease

Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.

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