Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

Zhu, Hongmei; Li, Jun; Cui, Chenchen; Song, Yujie; Ge, Hengtao; Hu, Linyong; Li, Qian; Jin, Yaping; Zhang, Yong

doi:10.1371/journal.pone.0156636

Cited by 12 publications

(9 citation statements)

References 40 publications

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“…This initial report presented the potential of the TALEN endonucleases for the introduction of desirable allelic introgressions into the genomes of farm animals. TALENs have also been applied in goats to target the BLG gene (Cui et al, 2015; Ge et al, 2016; Zhu et al, 2016b; Yuan et al, 2017). Furthermore, gene-edited goats that carry both BLG knockout and enriched expression of human lactoferrin (hLF) (Cui et al, 2015) or human α-lactalbumin (hLA) (Zhu et al, 2016b) have been generated using SCNT.…”

Section: Overview Of Genetic Modification and Relevant Biotechnologicmentioning

confidence: 99%

“…Furthermore, Zhu et al reported the generation of BLG knockout goats with enriched expression of hLA in milk, six live births were obtained, one was a biallelic targeted goat (which died after 1 month), and the other five goats were BLG hLA/+ . BLG expression in the milk of transgenic goats was reduced, while hLA was highly expressed compared to normal goats (Zhu et al, 2016b). Caprine MSTN knockout using TALENs has also been reported.…”

Section: Overview Of Genetic Modification and Relevant Biotechnologicmentioning

confidence: 99%

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Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

et al. 2019

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Sheep and goats are valuable livestock species that have been raised for their production of meat, milk, fiber, and other by-products. Due to their suitable size, short gestation period, and abundant secretion of milk, sheep and goats have become important model animals in agricultural, pharmaceutical, and biomedical research. Genome engineering has been widely applied to sheep and goat research. Pronuclear injection and somatic cell nuclear transfer represent the two primary procedures for the generation of genetically modified sheep and goats. Further assisted tools have emerged to enhance the efficiency of genetic modification and to simplify the generation of genetically modified founders. These tools include sperm-mediated gene transfer, viral vectors, RNA interference, recombinases, transposons, and endonucleases. Of these tools, the four classes of site-specific endonucleases (meganucleases, ZFNs, TALENs, and CRISPRs) have attracted wide attention due to their DNA double-strand break-inducing role, which enable desired DNA modifications based on the stimulation of native cellular DNA repair mechanisms. Currently, CRISPR systems dominate the field of genome editing. Gene-edited sheep and goats, generated using these tools, provide valuable models for investigations on gene functions, improving animal breeding, producing pharmaceuticals in milk, improving animal disease resistance, recapitulating human diseases, and providing hosts for the growth of human organs. In addition, more promising derivative tools of CRISPR systems have emerged such as base editors which enable the induction of single-base alterations without any requirements for homology-directed repair or DNA donor. These precise editors are helpful for revealing desirable phenotypes and correcting genetic diseases controlled by single bases. This review highlights the advances of genome engineering in sheep and goats over the past four decades with particular emphasis on the application of CRISPR/Cas9 systems.

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Section: Overview Of Genetic Modification and Relevant Biotechnologicmentioning

confidence: 99%

Section: Overview Of Genetic Modification and Relevant Biotechnologicmentioning

confidence: 99%

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

et al. 2019

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“…The emphasis on this study, however, was on the high level expression of human serum albumin for further purification from milk and not at using the BLG-free, human serum albumin-rich milk as food. Similarly, BLG knockout combining TALENs-mediated gene editing and SCNT was also performed in goats and resulted in targeted mono- and bi-allelic gene exchange of BLG against either human lactoferrin (hLf) or human alpha-lactalbumin (hLA) 7 , 8 . Biallelically targeted BLG hLf/hLf goats were shown to no longer produce BLG but high levels of hLf 8 , whereas monoallelically targeted BLG +/hLA goats exhibited slightly reduced BLG and high expression of hLA 7 .…”

Section: Introductionmentioning

confidence: 99%

Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin

Wei

Wagner

Maclean

et al. 2018

Sci Rep

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We applied precise zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows’ milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the two male calves. The third calf, a healthy female, had in addition to the expected nine bp deletion (81%), alleles with an in frame 21 bp deletion (<17%) at the target site. While her milk was free of any mature BLG, we detected low levels of a BLG variant derived from the minor deletion allele. This confirmed that the nine bp deletion genotype completely knocks out production of BLG. In addition, we showed that the LGB knockout animals are free of any TALEN-mediated off-target mutations or vector integration events using an unbiased whole genome analysis. Our study demonstrates the feasibility of generating precisely biallelically edited cattle by zygote-mediated editing for the safe production of hypoallergenic milk.

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“…The TALEN-mediated system was also used to knockout the BLG gene to reduce allergens and to create a knockin of human lactoferrin (hLF) for practical use as a bioreactor (Cui et al, 2015). Furthermore, targeting exon 1 of the BLG locus has been efficiently applied (Zhu et al, 2016). Goat MSTN gene editing has also been examined using the TALEN-mediated system (Yu et al, 2016).…”

Section: Goatmentioning

confidence: 99%

Recent progress toward precise genome editing in animals

2017

The Korean Society of Animal Breeding and Genomics

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The ability to edit a specific locus in a genome based on targeted DNA nucleases will greatly facilitate our understanding of the function of a gene in biological research, and have many practical uses in animal biotechnology and biomedicine. Recent advances, including clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), have substantially increased the efficiency of precise genome editing in a targeted manner. Therefore, this system has been successfully adopted for highly efficient genetic modification in diverse organisms, including animals, plants and humans, and is applicable to the efficient production of transgenic animals and model animals, as well as to breeding and research involving therapeutic potential. In addition, a base editing system has been recently developed and utilized for precise base conversion. It is composed of catalytically dead Cas9 (dCas9) and cytidine deaminase that enables both highly targeted single-base changes and local sequence diversification without double-stranded DNA cleavage. In this review, we introduce the development of genome editing technologies based on targeted DNA nucleases and the recently developed base editing system, and highlight successful research achievements indicating the potential use of programmable genome editing tools in fields of animal biotechnology.

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Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

Cited by 12 publications

References 40 publications

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era

Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin

Recent progress toward precise genome editing in animals

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