i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats

Takabayashi, Shuji; Aoshima, Takuya; Kabashima, Katsuya; Aoto, Kazushi; Ohtsuka, Masato

doi:10.1038/s41598-018-30137-x

Cited by 40 publications

(66 citation statements)

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“…Based on these results, Ohtsuka et al [81] renamed this modified technology, improved GONAD (i-GONAD). The i-GONAD was also proven to be effective in rats [82,83]. The detailed protocols for GONAD and i-GONAD have been reported by Gurumurthy et al [84,85].…”

Section: Gonad/i-gonad For Obtaining Ge Animalsmentioning

confidence: 92%

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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Section: Gonad/i-gonad For Obtaining Ge Animalsmentioning

confidence: 92%

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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“…Subsequently, another report on i ‐GONAD in rats was published by Takabayashi et al. (Takabayashi et al., ). Takabayashi et al.…”

Section: Potential Applications Of I‐gonadmentioning

confidence: 99%

“…Takabayashi et al. () successfully disrupted the wild‐type allele of Tyr locus in the BN strain (62% [8/13]) and rescued the mutation in LEW (5% [1/22]) and SD (5% [2/40]) strains. The authors also performed i ‐GONAD in hybrid embryos (derived from a cross between SD and BN strains) and obtained embryos with mutation in the paired box 6 ( Pax6 ) locus (50% [4/8]).…”

Section: Potential Applications Of I‐gonadmentioning

confidence: 99%

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

Ohtsuka

2019

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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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“…We re-named this improved technology as improved genome-editing via oviductal nucleic acid delivery ("improved GONAD (i-GONAD)"). i-GONAD is also demonstrated to be an effective tool for in vivo genome-editing in rats [20,21].…”

Section: Introductionmentioning

confidence: 99%

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice

Miyagasako

Takabayashi

Ohtsuka

et al. 2020

Cells

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Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4–1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4–1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called “sequential i-GONAD (si-GONAD).”

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i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats

Cited by 40 publications

References 40 publications

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

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

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice

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