<scp>CRISPR</scp>/Cas9‐mediated specific integration of <i>fat‐1</i> at the goat <i><scp>MSTN</scp></i> locus

Zhang, Ju; Cui, Meng-Lan; Nie, Yongwei; Dai, Bai; Li, Feiran; Liu, Dongjun; Liang, Hao; Cang, Ming

doi:10.1111/febs.14520

Cited by 36 publications

(12 citation statements)

References 41 publications

(44 reference statements)

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“…Further publications have also reported the generation of MSTN knockout sheep and goats via the CRISPR/Cas9 system (see Table 3 ). Among these, an interesting report described the generation of a goat kid carrying simultaneous MSTN knockout and fat1 knockin using CRISPR/Cas9 combined with SCNT (Zhang et al, 2018a). The efficiency of simultaneous targeting was 25.6% (40/156) in goat fibroblasts.…”

Section: Applications Of Crispr/cas9 In Sheep and Goatsmentioning

confidence: 99%

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: Applications Of Crispr/cas9 In Sheep and Goatsmentioning

confidence: 99%

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

et al. 2019

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“…However, these studies almost exclusively integrated one foreign gene, and only one trait was improved for genetically modified pigs. This is far from sufficient for genetic breeding [ 36 ], xenotransplantation [ 37 ], and other transgenic research, which require the improvement of multiple simultaneously. Therefore, it is particularly important to integrate multiple foreign genes into the same site.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus

You

et al. 2021

Genes

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Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.

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“…The protein bands were visualized with a Pierce ECL western blotting substrate (Thermo Fisher Scientific, Munich, Germany), using the Tanon 5200 (Tanon, Shanghai, China) detection system. GAPDH was used as the loading control [31,34].…”

Section: Western Blot Analysismentioning

confidence: 99%

The Overexpression of Tβ4 in the Hair Follicle Tissue of Alpas Cashmere Goats Increases Cashmere Yield and Promotes Hair Follicle Development

Dai

Liang

Guo

et al. 2019

Animals

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Increased cashmere yield and improved quality are some goals of cashmere goat breeding. Thymosin beta-4 (Tβ4) plays a key role in the growth and development of hair follicles. For the past ten years, we have evaluated the role of Tβ4 by establishing a flock of 15 cashmere goats that specifically overexpress the Tβ4 gene in the hair follicles. These Tβ4 overexpression (Tβ4-OE) cashmere goats had more secondary hair follicles than the WT goats and produced more cashmere. Meanwhile, combined analysis of the skin transcriptome and proteome in cashmere goats suggested that Tβ4 may affect hair growth by interacting with keratin type II cytoskeletal 4 epidermal (KRT4) to mediate the extracellular signal-regulated protein kinase (ERK) signaling pathway, thereby promoting the development of secondary hair follicles, and consequently, increasing cashmere yield. Thus, the specific overexpression of Tβ4 in the hair follicles of cashmere goats effectively increased the cashmere yield.

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CRISPR/Cas9‐mediated specific integration of fat‐1 at the goat MSTN locus

Cited by 36 publications

References 41 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

CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus

The Overexpression of Tβ4 in the Hair Follicle Tissue of Alpas Cashmere Goats Increases Cashmere Yield and Promotes Hair Follicle Development

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