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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 Biology2.94
· 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)
Cite
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)
Cite
References23
Newest
Published on Jun 1, 2016in Development5.76
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...
Published on Dec 1, 2015in Genome Biology14.03
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...
Published on Sep 1, 2015in Scientific Reports4.01
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
Published on Jun 8, 2015in PLOS ONE2.78
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...
Published on Jun 1, 2015in Genetics3.56
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...
Published on May 1, 2015in Scientific Reports4.01
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...
Published on May 1, 2015in Scientific Reports4.01
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...
Published on Sep 1, 2014in Developmental Biology2.94
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...
Published on Aug 1, 2014in Mammalian Genome2.34
Seiya Mizuno10
Estimated H-index: 10
(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...
Published on Jan 1, 2014in Genome Research9.94
Young Hoon Sung10
Estimated H-index: 10
(Yonsei University),
Jong Min Kim1
Estimated H-index: 1
(SNU: Seoul National University)
+ 9 AuthorsJin-Soo Kim52
Estimated H-index: 52
(SNU: Seoul 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...
Cited By47
Newest
Published on Dec 1, 2019in Scientific Reports4.01
Takayuki Sakurai15
Estimated H-index: 15
(Shinshu University),
Akiko Kamiyoshi12
Estimated H-index: 12
(Shinshu University)
+ 3 AuthorsTakayuki Shindo32
Estimated H-index: 32
(Shinshu University)
We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as ‘negative’ under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstrea...
Published on Feb 6, 2019in Scientific Reports4.01
Sho Yoshimatsu1
Estimated H-index: 1
(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...
Published on Dec 1, 2019in Scientific Reports4.01
Masahito Watanabe11
Estimated H-index: 11
(International Institute of Minnesota),
Kazuaki Nakano12
Estimated H-index: 12
(Meiji University)
+ 12 AuthorsSumiyo Morita16
Estimated H-index: 16
(Gunma University)
To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced ...
Published on Sep 3, 2019in Transgenic Research1.82
Charles-Etienne Dumeau (ETH Zurich), Asun Monfort (ETH Zurich)+ 3 AuthorsAnton Wutz19
Estimated H-index: 19
(ETH Zurich)
CRISPR-associated (Cas) nucleases are established tools for engineering of animal genomes. These programmable RNA-guided nucleases have been introduced into zygotes using expression vectors, mRNA, or directly as ribonucleoprotein (RNP) complexes by different delivery methods. Whereas microinjection techniques are well established, more recently developed electroporation methods simplify RNP delivery but can provide less consistent efficiency. Previously, we have designed Cas12a-crRNA pairs to in...
Published on Aug 1, 2019in Current Opinion in Biotechnology8.08
Hideki Ukai8
Estimated H-index: 8
(UTokyo: University of Tokyo),
Hideki Ukai (UTokyo: University of Tokyo)+ 0 AuthorsHiroki 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...
Published on Jul 24, 2019in Nature Protocols11.33
Channabasavaiah B. Gurumurthy15
Estimated H-index: 15
(UNMC: University of Nebraska Medical Center),
Masahiro Sato30
Estimated H-index: 30
(Kadai: Kagoshima University)
+ 9 AuthorsShinichi Nakagawa (Hokkaido University)
Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRN...
Published on Apr 26, 2019in Biology of Reproduction2.96
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)
Published on 2019in Developmental Cell9.19
Masakazu Hashimoto7
Estimated H-index: 7
(Osaka University),
Hiroshi Sasaki51
Estimated H-index: 51
(Osaka University)
Summary The epiblast is a pluripotent cell population first formed in preimplantation embryos, and its quality is important for proper development. Here, we examined the mechanisms of epiblast formation and found that the Hippo pathway transcription factor TEAD and its coactivator YAP regulate expression of pluripotency factors. After specification of the inner cell mass, YAP accumulates in the nuclei and activates TEAD. TEAD activity is required for strong expression of pluripotency factors and...
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