Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing

Teixeira, Marie; Py, Bénédicte F.; Bosc, Christophe; Laubreton, Daphné; Moutin, Marie‐Jo; Marvel, Jacqueline; Flamant, Frédéric; Markossian, Suzy

doi:10.1038/s41598-017-18826-5

Cited by 65 publications

(56 citation statements)

References 34 publications

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“…Although it remains interesting whether the nature of the sgRNA or traces from its generation are responsible for the embryo toxicity our data validate synthetic pgRNAs as less harmful during electroporation. While our manuscript was in preparation another group independently used synthetic pgRNAs for successful generation of CRISPR/Cas9-mediated mouse mutants via electroporation [18]. Although they did not compare pgRNA and sgRNA side-by-side their data nicely confirm our finding that synthetic pgRNA is indeed an efficient and safe alternative to conventional sgRNA.…”

Section: Discussionmentioning

confidence: 59%

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

Tröder

Ebert

Butt

et al. 2018

Preprint

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Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6N mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring.Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Section: Discussionmentioning

confidence: 59%

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

Tröder

Ebert

Butt

et al. 2018

Preprint

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“…Since Kaneko et al [36] first applied this technology to zygotes for producing GE animals, successful genome editing of mice [37][38][39][40][41][42] using this technology has become possible. Compared to the previously described microinjection-based technique, several zygotes (30 to 50) can be simultaneously genome edited using a square pulse generator (electroporator), a technique that does not require expensive micromanipulator systems.…”

Section: Ep Techniquementioning

confidence: 99%

“…Requires ICSI and egg transfer to allow further development of the treated embryos [12][13][14][15][36][37][38][39][40][41][42][43][44][45]…”

Section: Ex Vivomentioning

confidence: 99%

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Takabayashi

Akasaka

Nakamura

2020

Cells

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The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (i-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research.

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“…The success of this technology was subsequently reported by Kaneko et al. and other researchers using mice (Chen, Lee, Lee, Modzelewski, & He, ; Hashimoto, Yamashita, & Takemoto, ; Kaneko & Mashimo, ; Qin et al., ; Teixeira et al., ; Tröder et al., ). Although this approach simplified the process of gene delivery into embryos, it still requires ex vivo handling of embryos, such as embryo collection, short periods of embryo culture, and subsequent ET (Figure ; Table ).…”

Section: Historical Backgroundmentioning

confidence: 84%

i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

Ohtsuka

2019

Dev Growth Differ

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The recent development of genome editing technologies has enabled the creation of genome‐edited animals, with alterations at the desired target locus. The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for this purpose because it is simpler and more efficient than other genome editing technologies. The conventional methods for creation of genome‐edited animals involve ex vivo handling of embryos (zygotes) for microinjection or in vitro electroporation. However, this process is laborious and time‐consuming, and relatively large numbers of animals are used. Furthermore, these methods require specialized skills for handling embryos. In 2015, we reported a novel method for the creation of genome‐edited animals without ex vivo handling of embryos. The technology known as Genome‐editing via Oviductal Nucleic Acids Delivery (GONAD) involved intraoviductal instillation of genome editing components into a pregnant female and subsequent in vivo electroporation of an entire oviduct. The genome editing components present in the oviductal lumen are transferred to preimplantation embryos in situ for introducing insertion or deletion (indel) mutations at the desired loci. This technology was further improved by optimizing several parameters to develop improved GONAD (i‐GONAD) for the efficient generation of mutant or knock‐in animals. In this review, we discuss the historical background, potential applications, advantages, and future challenges of GONAD/i‐GONAD technology.

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Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing

Cited by 65 publications

References 34 publications

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

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