CLICK: one-step generation of conditional knockout mice

Miyasaka, Yoshiki; Uno, Yoshihiro; Yoshimi, Kazuto; Kunihiro, Yayoi; Yoshimura, Takuji; Tanaka, Tomohiro; Ishikubo, Harumi; Hiraoka, Yuichi; Takemoto, Norihiko; Tanaka, Takao; Ooguchi, Yoshihiro; Skehel, Paul; Aida, Tomomi; Takeda, Junji; Mashimo, Tomoji

doi:10.1186/s12864-018-4713-y

Cited by 85 publications

(88 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, Miyasaka et al. succeeded in introducing large fragment into rodent zygotes by electroporation of long single-strand donor DNA without micromanipulation; however, the donor DNA were up to 1.1 kb, including homology arms ( Miyasaka et al., 2018 ). Pronuclear injection is a highly skilled technique and relatively difficult in non-rodent mammals.…”

Section: Discussionmentioning

confidence: 99%

Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector

et al. 2018

View full text Add to dashboard Cite

SummaryIntra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation.

show abstract

Section: Discussionmentioning

confidence: 99%

Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector

et al. 2018

View full text Add to dashboard Cite

show abstract

“…The desire to apply CRISPR/Cas9 for the targeted insertion of transgenes is reflected in the profusion of methods directed towards this purpose [63,108,110,112,186,187]. Each method was successfully used to engineer mouse and rat genomes (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Principles of Genetic Engineering

Lanigan

Kopera

Saunders

2020

Genes

View full text Add to dashboard Cite

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

show abstract

“…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

View full text Add to dashboard Cite

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.

show abstract

CLICK: one-step generation of conditional knockout mice

Cited by 85 publications

References 21 publications

Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector

Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector

Principles of Genetic Engineering

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

Contact Info

Product

Resources

About