Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

Dumeau, Charles-Étienne; Monfort, Asun; Kissling, Lucas; Swarts, Daan C.; Jinek, Martin; Wutz, Anton

doi:10.1007/s11248-019-00168-9

Cited by 22 publications

(19 citation statements)

References 33 publications

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“…We recently developed a new method, named TAKE, that could produce genome-edited animals by electroporation instead of the microinjection method [ 5 , 6 , 7 ]. Many kinds of knockout and knock-in mice and rats have already been produced by TAKE method using ZFN, TALEN, CRISPR-Cas system [ 12 , 13 , 14 , 15 , 16 , 17 ], and other nucleases [ 18 ]. Furthermore, this method has widely been applied for the production of genome-edited strains in other animals [ 19 , 20 ].…”

Section: Discussionmentioning

confidence: 99%

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

Wake

Kaneko

2020

Journal of Reproduction and Development

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Genome editing technology contributes to the quick and highly efficient production of genetically engineered animals. These animals are helpful in clarifying the mechanism of human disease. Recently, a new electroporation technique (TAKE: Technique for animal knockout system by electroporation) was developed to produce genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. The aim of this study was to increase the efficiency of production of genome-edited animals using the TAKE method. In the conventional protocol, it was difficult to confirm the introduction of nucleases into embryos and energization during operation. Using only embryos that introduced nucleases for embryo transfer, it will lead to increased efficiency in the production of genome-edited animals. This study examined the visualization in the introduction of nucleases into the embryos by using nucleases fluorescent labeled with ATTO-550. The embryos were transfected with Cas9 protein and fluorescent labeled dual guide RNA (mixture with crRNA and tracrRNA with ATTO-550) targeted tyrosinase gene by the TAKE method. All embryos that survived after electroporation showed fluorescence. Of these embryos with fluorescence, 43.7% developed to morphologically normal offspring. In addition, 91.7% of offspring were edited by the tyrosinase gene. This study is the first to demonstrate that the introduction of nucleases into embryos by the TAKE method could be visualized using fluorescent-labeled nucleases. This improved TAKE method can be used to produce genome-edited animals and confirm energization during operation.

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Section: Discussionmentioning

confidence: 99%

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

Wake

Kaneko

2020

Journal of Reproduction and Development

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“…Another study published in 2019 utilized Cas12a instead of Cas9 as the nuclease, and targeted three different genes with electroporation. The authors found knockout mutation rates in mouse embryos ranged from 34 to 70% (Dumeau et al, 2019). Unfortunately, mosaicism rates were not studied.…”

Section: Electroporation-mediated Knockoutsmentioning

confidence: 99%

Electroporation-Mediated Genome Editing of Livestock Zygotes

Lin

Eenennaam

2021

Front. Genet.

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The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

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“…Of the challenges and approaches of delivering CRISPR, it was pointed out [ 18 , 51 ] that although the present genome engineering is in favor of CRISPR tools, TALENs may still be of a primary choice in certain experimental species. For example, TALENs have been utilized in targeted genomic editing in Xenopus tropicalis by knocking-out Klf4 [ 49 , 50 ] or thyroid hormone receptor α [ 23 ].…”

Section: Gene Deliverymentioning

confidence: 99%

“…To deliver the carrying DNA sequence to target cells, non-viral techniques such as electroporation, lipofection, and microinjection can also be used [ 18 ]. In addition, these techniques also reduce off-target cleavages problems.…”

Section: Gene Deliverymentioning

confidence: 99%

The genome editing revolution: review

Khalil

2020

Journal of Genetic Engineering and Biotechnology

156

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Background Development of efficient strategies has always been one of the great perspectives for biotechnologists. During the last decade, genome editing of different organisms has been a fast advancing field and therefore has received a lot of attention from various researchers comprehensively reviewing latest achievements and offering opinions on future directions. This review presents a brief history, basic principles, advantages and disadvantages, as well as various aspects of each genome editing technology including the modes, applications, and challenges that face delivery of gene editing components. Main body Genetic modification techniques cover a wide range of studies, including the generation of transgenic animals, functional analysis of genes, model development for diseases, or drug development. The delivery of certain proteins such as monoclonal antibodies, enzymes, and growth hormones has been suffering from several obstacles because of their large size. These difficulties encouraged scientists to explore alternative approaches, leading to the progress in gene editing. The distinguished efforts and enormous experimentation have now been able to introduce methodologies that can change the genetic constitution of the living cell. The genome editing strategies have evolved during the last three decades, and nowadays, four types of “programmable” nucleases are available in this field: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system. Each group has its own characteristics necessary for researchers to select the most suitable method for gene editing tool for a range of applications. Genome engineering/editing technology will revolutionize the creation of precisely manipulated genomes of cells or organisms in order to modify a specific characteristic. Of the potential applications are those in human health and agriculture. Introducing constructs into target cells or organisms is the key step in genome engineering. Conclusions Despite the success already achieved, the genome editing techniques are still suffering certain difficulties. Challenges must be overcome before the full potential of genome editing can be realized.

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Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1

Cited by 22 publications

References 33 publications

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

Electroporation-Mediated Genome Editing of Livestock Zygotes

The genome editing revolution: review

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