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

Published on Oct 1, 2016in Developmental Biology 3.26
· DOI :10.1016/j.ydbio.2016.07.017
Masakazu Hashimoto7
Estimated H-index: 7
(Osaka University),
Yukiko Yamashita2
Estimated H-index: 2
(University of Tokushima),
Tatsuya Takemoto13
Estimated H-index: 13
(University of Tokushima)
Abstract
Abstract 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.
  • References (23)
  • Citations (47)
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References23
Newest
Published on Jun 1, 2016in Development 5.41
Alexa Burger7
Estimated H-index: 7
(UZH: University of Zurich),
Helen Lindsay12
Estimated H-index: 12
(Swiss Institute of Bioinformatics)
+ 9 AuthorsMartin Jinek26
Estimated H-index: 26
(UZH: University of Zurich)
CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro -assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zeb...
66 Citations Source Cite
Published on Dec 1, 2015in Genome Biology 13.21
Tomomi Aida11
Estimated H-index: 11
(Tokyo Medical and Dental University),
Keiho Chiyo1
Estimated H-index: 1
(Tokyo Medical and Dental University)
+ 7 AuthorsKohichi Tanaka51
Estimated H-index: 51
(Tokyo Medical and Dental University)
Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA...
122 Citations Source Cite
Published on Sep 1, 2015in Scientific Reports 4.12
Masakazu Hashimoto7
Estimated H-index: 7
,
Tatsuya Takemoto13
Estimated H-index: 13
Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing
92 Citations Source Cite
Published on Jun 8, 2015in PLOS ONE 2.77
Daniel Oliver5
Estimated H-index: 5
(UNR: University of Nevada, Reno),
Shuiqiao Yuan11
Estimated H-index: 11
(UNR: University of Nevada, Reno)
+ 1 AuthorsWei Yan44
Estimated H-index: 44
(UNR: University of Nevada, Reno)
Genome editing technologies, especially the Cas9/CRISPR system, have revolutionized biomedical research over the past several years. Generation of novel alleles has been simplified to unprecedented levels, allowing for rapid expansion of available genetic tool kits for researchers. However, the issue of genotypic mosaicism has become evident, making stringent analyses of the penetrance of genome-edited alleles essential. Here, we report that founder mice, derived from pronuclear injection of ZFN...
19 Citations Source Cite
Published on Jun 1, 2015in Genetics 4.08
Wenning Qin3
Estimated H-index: 3
,
Stephanie L. Dion2
Estimated H-index: 2
+ 7 AuthorsHaoyi Wang24
Estimated H-index: 24
(CAS: Chinese Academy of Sciences)
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 zygo...
87 Citations Source Cite
Published on May 1, 2015in Scientific Reports 4.12
Akihiro Yasue10
Estimated H-index: 10
,
Silvia Naomi Mitsui2
Estimated H-index: 2
+ 6 AuthorsEiji Tanaka59
Estimated H-index: 59
Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In ad...
40 Citations Source Cite
Published on May 1, 2015in Scientific Reports 4.12
Takehito Kaneko20
Estimated H-index: 20
,
Tetsushi Sakuma31
Estimated H-index: 31
+ 1 AuthorsTomoji Mashimo27
Estimated H-index: 27
Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into i...
64 Citations Source Cite
Published on Sep 1, 2014in Developmental Biology 3.26
Shuo Ting Yen1
Estimated H-index: 1
(BCM: Baylor College of Medicine),
Min Zhang12
Estimated H-index: 12
(BCM: Baylor College of Medicine)
+ 6 AuthorsRichard R. Behringer97
Estimated H-index: 97
Abstract Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene ( Tyr ) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Ty...
127 Citations Source Cite
Published on Aug 1, 2014in Mammalian Genome 2.69
Seiya Mizuno9
Estimated H-index: 9
(University of Tsukuba),
Tra Thi Huong Dinh3
Estimated H-index: 3
(University of Tsukuba)
+ 8 AuthorsFumihiro Sugiyama31
Estimated H-index: 31
(University of Tsukuba)
Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constru...
43 Citations Source Cite
Published on Jan 1, 2014in Genome Research 10.10
Young Hoon Sung10
Estimated H-index: 10
(Yonsei University),
Jong Min Kim1
Estimated H-index: 1
(SNU: Seoul National University)
+ 9 AuthorsCheol-Hee Kim36
Estimated H-index: 36
(CNU: Chungnam National University)
RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the m...
173 Citations Source Cite
Cited By47
Newest
Published on Feb 6, 2019in Scientific Reports 4.12
Sho Yoshimatsu (Keio: Keio University), Junko Okahara5
Estimated H-index: 5
(Central Institute for Experimental Animals)
+ 6 AuthorsHideyuki Okano110
Estimated H-index: 110
(Keio: Keio University)
Genome editing technology greatly facilitates the genetic modification of various cells and animals. The common marmoset (Callithrix jacchus), a small non-human primate which exhibits high reproductive efficiency, is a widely used animal model in biomedical research. Developing genome editing techniques in the common marmoset will further enhance its utility. Here, we report the successful establishment of a knock-in (KI) method for marmoset embryonic stem cells (ESCs), which is based on the CRI...
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Published on Dec 1, 2019in Scientific Reports 4.12
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Published on Aug 1, 2019in Current Opinion in Biotechnology 8.38
Hideki Ukai8
Estimated H-index: 8
(UTokyo: University of Tokyo),
Kenta Sumiyama24
Estimated H-index: 24
,
Hiroki R. Ueda43
Estimated H-index: 43
(UTokyo: University of Tokyo)
Systems-biological approaches, such as comprehensive identification and analysis of system components and networks, are necessary to understand design principles of human physiology and pathology. Although reverse genetics using mouse models have been used previously, it is a low throughput method because of the need for repetitive crossing to produce mice having all cells of the body with knock-out or knock-in mutations. Moreover, there are often issues from the interspecific gap between humans...
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Published on Apr 26, 2019in Biology of Reproduction 3.18
Deqiang Miao (WSU: Washington State University), Mariana Ianello Giassetti (WSU: Washington State University)+ 2 AuthorsJon M. Oatley29
Estimated H-index: 29
(WSU: Washington State University)
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Published on Jun 3, 2019in Nature Medicine 32.62
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Published on Jan 31, 2019in National Science Review 9.41
Jianguo Zhao (CAS: Chinese Academy of Sciences), Liangxue Lai (Guangzhou Institutes of Biomedicine and Health)+ 1 AuthorsQi Zhou51
Estimated H-index: 51
(CAS: Chinese Academy of Sciences)
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Published on May 1, 2019in Congenital Anomalies 1.15
Hideyo Ohuchi40
Estimated H-index: 40
(Okayama University),
Keita Sato5
Estimated H-index: 5
(Okayama University)
+ 2 AuthorsTetsuya Bando10
Estimated H-index: 10
(Okayama University)
2 Citations Source Cite