Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs

Sheets, Timothy P.; Park, Chi-Hun; Park, Ki‐Eun; Powell, Anne M.; Donovan, David M.; Telugu, Bhanu P.

doi:10.3390/ijms17122031

Cited by 41 publications

(25 citation statements)

References 26 publications

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“…Furthermore, GE pigs could be efficiently produced by in vitro EP in the presence of RNP [68]. Sheets et al [82] produced GE fetuses by cytoplasmic MI of RNP into oocytes reconstituted with intact cells using the SCNT technology. By this treatment, they reported highly efficient (100%) generation of bi-allelic modification in the resultant cloned fetuses.…”

Section: History Of Ge In Pigsmentioning

confidence: 99%

“…For example, in mice, delivering RNPs into zygotes causes rapid genome editing in the target locus, which also maximizes efficiency while minimizing mosaicism [110][111][112]. Indeed, Sheets et al [82] demonstrated that after MI with RNP, 100% of piglets produced had the bi-allelic KO genotype.…”

Section: MImentioning

confidence: 99%

“…Although direct modification of zygotic genomes provides some advantages, SCNT also provides a significant advantage by permitting the isolation of cells containing precise modifications before the expense of animal production is incurred. As mentioned previously, Sheets et al [82] successfully produced genome-edited cloned pigs by combining SCNT with CRISPR/Cas9 MI, which is beneficial for researchers as they do not need to manage a founder herd, and can eliminate the need for laborious in vitro culture and screening. In this study, all (6/6) of the resultant clone fetuses exhibited 100% bi-allelic modification.…”

Section: After Scntmentioning

confidence: 99%

See 2 more Smart Citations

Recent Advance in Genome Editing-Based Gene Modification in Pigs

Miyoshi¹,

Kawaguchi²,

Inada³

et al. 2020

Reproductive Biology and Technology in Animals

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Recently, a series of genome editing technologies including ZFNs, TALENs, and CRISPR/Cas9 systems have enabled gene modification in the endogenous target genes of various organisms including pigs, which are important for agricultural and biomedical research. Owing to its simple application for gene knockout and ease of use, the CRISPR/Cas9 is now in common use worldwide. The most important aspect of this process is the selection of the method used to deliver genome editing components to embryos. In earlier stages, zygote microinjection of these components [single guide RNA (sgRNA) + DNA/mRNA for Cas9] into the cytoplasm and/ or nuclei of a zygote has been frequently employed. However, this method is always associated with the generation of mosaic embryos in which genome-edited and unedited cells are mixed together. To avoid this mosaic issue, in vitro electroporation of zygotes in the presence of sgRNA mixed with Cas9 protein, referred to as a ribonucleoprotein (RNP), is now in frequent use. This review provides a historical background of the production of genome-edited pigs and also presents current research concerning how genome editing is induced in somatic cell nuclear transferderived embryos that have been reconstituted with normal nuclei. GE piglets have been produced using SCNT of genome-edited cells, or direct microinjection of genome-editing components (including engineered endonucleases) into the cytoplasm of zygotes, as described below in more detail. Background of second-generation genome editingAs mentioned above, site-specific engineered nucleases are used in these genome-editing techniques. ZFNs, TALENs, and CRISPR/Cas9 can all bind to DNA and induce DSB, which triggers endogenous DNA repair. If the template DNA is absent, the DSB is repaired via the NHEJ pathway where insertion or deletion of nucleotides (hereinafter called "indels") can happen in the cleaved area. These indels often cause frameshift of the amino acid sequence, leading to the generation of abnormal proteins or formation of a premature stop codon leading to cessation of protein synthesis. If template DNA homologous to the target site is present, it is inserted into the cleaved area via a site-specific HR event which is called HDR. Generally, NHEJ occurs in cells independent of its cell cycle, but HDR occurs primarily in dividing cells [30].The ZFN technique uses the ZF protein (which binds to the target DNA) and the endonuclease Fok I (which cleaves DNA) [31]. ZF protein has several protein motifs capable of recognizing specific sequences of three nucleotides and binding to them. Notably, Urnov et al. [32] first demonstrated that ZFN is effective to induce DNA editing at the endogenous target gene in mammalian cells. Its targeting efficiency was over 18% in the absence of drug selection, which is ~1000-fold higher than that achieved by traditional gene targeting.The TALEN technique uses proteins, termed transcription activator-like effectors (TALEs), which contain 33-35 amino acid repeats that flank a central DNA binding region (a...

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Section: History Of Ge In Pigsmentioning

confidence: 99%

Section: MImentioning

confidence: 99%

Section: After Scntmentioning

confidence: 99%

See 1 more Smart Citation

Recent Advance in Genome Editing-Based Gene Modification in Pigs

Miyoshi¹,

Kawaguchi²,

Inada³

et al. 2020

Reproductive Biology and Technology in Animals

View full text Add to dashboard Cite

show abstract

“…Litter sizes and viability are good with this approach, and while early demonstrations had relatively low editing frequency, evolution of the tools and refinement in delivery has resulted in substantive improvements [10,11] . A combination of SCNT followed by zygote microinjection with Cas9 ribonucleoprotein (Cas9 protein pre-complexed with sgRNA) has also been shown to be a very efficient strategy for generation of genome edited pigs [12] .…”

Section: Application In Livestockmentioning

confidence: 99%

Livestock breeding for the 21st century: the promise of the editing revolution

Proudfoot¹,

McFarlane²,

Whitelaw³

et al. 2020

Front. Agr. Sci. Eng.

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In recent years there has been a veritable explosion in the use of genome editors to create sitespecific changes, both in vitro and in vivo, to the genomes of a multitude of species for both basic research and biotechnology. Livestock, which form a vital component of most societies, are no exception. While selective breeding has been hugely successful at enhancing some production traits, the rate of progress is often slow and is limited to variants that exist within the breeding population. Genome editing provides the potential to move traits between breeds, in a single generation, with no impact on existing productivity or to develop de novo phenotypes that tackle intractable issues such as disease. As such, genome editors provide huge potential for ongoing livestock development programs in light of increased demand and disease challenge. This review will highlight some of the more notable agricultural applications of this technology in livestock.

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“…; Wang, Du, et al., ; Wang, Zhou, et al., ; Whitworth et al., ; Yu et al., ), sheep (Crispo et al., ; Wang, Niu, et al., ; Zhang et al., ), goat (Guo et al., ; Wang, Yu, et al., ) and cattle (Bevacqua et al., ). CRISPR can be also delivered as ribonucleoprotein (Park, Powell, et al., ; Sheets et al., ), with the advantage that it acts faster than RNA delivery, as it does not require the generation of Cas9 protein by the zygote. The components can be also delivered as plasmid (Chuang et al., ; Honda et al., ; Petersen et al., ), but this is the slowest way, as Cas9 needs to be transcribed and translated, and it entails a prolonged presence of CRISPR components that favours the appearance of off‐target effects (OTEs).…”

Section: State Of the Artmentioning

confidence: 99%

CRISPR is knocking on barn door

Lamas-Toranzo

Guerrero-Sánchez

Miralles-Bover

et al. 2017

Reprod Domestic Animals

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Genome modification at specific loci in livestock species was only achievable by performing homologous recombination in somatic cells followed by somatic cell nuclear transfer. The difficulty and inefficiency of this method have slowed down the multiple applications of genome modification in farm animals. The discovery of site-specific endonucleases has provided a different and more direct route for targeted mutagenesis, as these enzymes allow the ablation (KO) or insertion (KI) of specific genomic sequences on a single step, directly applied to zygotes. Clustered regularly interspaced short palindromic repeats (CRISPR), the last site-specific endonuclease to be developed, is a RNA-guided endonuclease, easy to engineer and direct to a given target site. This technology has been successfully applied to rabbits, swine, goats, sheep and cattle, situating genome editing in livestock species at an attainable distance, thereby empowering scientist to develop a myriad of applications. Genetically modified livestock animals can be used as biomodels to study human or livestock physiology and disease, as bioreactors to produce complex proteins, or as organ donors for transplantation. Specifically on livestock production, genome editing in farm animals may serve to improve productive genetic traits, to improve various animal products, to confer resistance to diseases or to minimize the environmental impact on farming. In this review, we provide an overview of the current methods for site-specific genome modification in livestock species, discuss potential and already developed applications of genome edition in farm animals and debate about the possibilities for approval of products derived from gene-edited animals for human consumption.

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Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs

Cited by 41 publications

References 26 publications

Recent Advance in Genome Editing-Based Gene Modification in Pigs

Recent Advance in Genome Editing-Based Gene Modification in Pigs

Livestock breeding for the 21st century: the promise of the editing revolution

CRISPR is knocking on barn door

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