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  • 1
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    Online Resource
    Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep
    Springer Science and Business Media LLC ; 2016
    In:  Scientific Reports Vol. 6, No. 1 ( 2016-08-26)
    Details
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 6, No. 1 ( 2016-08-26)
    Abstract: The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes ( MSTN , ASIP and BCO2 ). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27–33% in generated lambs and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, prescreening for the off-target effect of sgRNAs was performed on fibroblasts before microinjection, to ensure that no detectable off-target mutations from founder animals existed. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by simultaneously targeting multiple genes that are responsible for economically significant traits.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    URL: Article
    DOI: 10.1038/srep32271
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2016
    detail.hit.zdb_id: 2615211-3
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  • 2
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    Efficient generation of goats with defined point mutation (I397V) in GDF9 through CRISPR/Cas9
    CSIRO Publishing ; 2018
    In:  Reproduction, Fertility and Development Vol. 30, No. 2 ( 2018), p. 307-
    Details
    In: Reproduction, Fertility and Development, CSIRO Publishing, Vol. 30, No. 2 ( 2018), p. 307-
    Abstract: The recent emergence of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has attracted significant attention for its potential to improve traits of agricultural importance. However, most applications in livestock species to date have depended on aberrant DNA repair to generate frameshifting indels. Whether this genomic engineering technique involving homology-dependent repair (HDR) can be used to introduce defined point mutations has been less explored. Previously, we reported a G→A point mutation (g.231A 〉 G, p.Val397Ile) in the growth differentiation factor 9 (GDF9) gene that has a large effect on the litter size of cashmere goats. In the present study we report that by co-injecting synthesised RNAs and single-stranded oligo deoxynucleotide (ssODN) donor sequences into goat zygotes, we successfully introduced defined point mutations resulting in single amino acid substitutions in the proteins as expected. The efficiency of this precise single-nucleotide substitution in newborn kids was as high as 24% (4/17), indicating that ssODN-directed HDR via zygote injection is efficient at introducing point mutations in the goat genome. The findings of the present study further highlight the complex genome modifications facilitated by the CRISPR/Cas9 system, which is able to introduce defined point mutations. This represents a significant development for the improvement of reproduction traits in goats, as well as for validating the roles of specific nucleotides in functional genetic elements in large animals.
    Type of Medium: Online Resource
    ISSN: 1031-3613
    URL: Article
    DOI: 10.1071/RD17068
    Language: English
    Publisher: CSIRO Publishing
    Publication Date: 2018
    SSG: 12
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  • 3
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    Base pair editing in goat: nonsense codon introgression into FGF 5 results in longer hair
    Wiley ; 2019
    In:  The FEBS Journal Vol. 286, No. 23 ( 2019-12), p. 4675-4692
    Details
    In: The FEBS Journal, Wiley, Vol. 286, No. 23 ( 2019-12), p. 4675-4692
    Abstract: The ability to alter single bases without homology directed repair ( HDR ) of double‐strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sg RNA s to induce nonsense codons (C‐to‐T transitions) at four target sites in caprine FGF 5 , which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor ( BE 3) plasmid and four different sg RNA s into caprine fibroblasts were ineffective in altering FGF 5 . In contrast, all five progenies produced from microinjected single‐cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE 3 to make single base changes varied considerably based on sg RNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF 5 expression. PCR amplicon and whole‐genome resequencing analyses for off‐target changes caused by BE 3 were low at a genome‐wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single‐cell embryos. Our results support further optimization of BE s for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation.
    Type of Medium: Online Resource
    ISSN: 1742-464X , 1742-4658
    URL: Issue
    URL: Article
    DOI: 10.1111/febs.v286.23
    DOI: 10.1111/febs.14983
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 2172518-4
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  • 4
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    Markhor-derived Introgression of a Genomic Region Encompassing PAPSS2 Confers High-altitude Adaptability in Tibetan Goats
    Oxford University Press (OUP) ; 2022
    In:  Molecular Biology and Evolution Vol. 39, No. 12 ( 2022-12-05)
    Details
    In: Molecular Biology and Evolution, Oxford University Press (OUP), Vol. 39, No. 12 ( 2022-12-05)
    Abstract: Understanding the genetic mechanism of how animals adapt to extreme conditions is fundamental to determine the relationship between molecular evolution and changing environments. Goat is one of the first domesticated species and has evolved rapidly to adapt to diverse environments, including harsh high-altitude conditions with low temperature and poor oxygen supply but strong ultraviolet radiation. Here, we analyzed 331 genomes of domestic goats and wild caprid species living at varying altitudes (high & gt; 3000 m above sea level and low & lt; 1200 m), along with a reference-guided chromosome-scale assembly (contig-N50: 90.4 Mb) of a female Tibetan goat genome based on PacBio HiFi long reads, to dissect the genetic determinants underlying their adaptation to harsh conditions on the Qinghai-Tibetan Plateau (QTP). Population genomic analyses combined with genome-wide association studies (GWAS) revealed a genomic region harboring the 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2) gene showing strong association with high-altitude adaptability (PGWAS = 3.62 × 10−25) in Tibetan goats. Transcriptomic data from 13 tissues revealed that PAPSS2 was implicated in hypoxia-related pathways in Tibetan goats. We further verified potential functional role of PAPSS2 in response to hypoxia in PAPSS2-deficient cells. Introgression analyses suggested that the PAPSS2 haplotype conferring the high-altitude adaptability in Tibetan goats originated from a recent hybridization between goats and a wild caprid species, the markhor (Capra falconeri). In conclusion, our results uncover a hitherto unknown contribution of PAPSS2 to high-altitude adaptability in Tibetan goats on QTP, following interspecific introgression and natural selection.
    Type of Medium: Online Resource
    ISSN: 0737-4038 , 1537-1719
    URL: Article
    DOI: 10.1093/molbev/msac253
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2022
    detail.hit.zdb_id: 2024221-9
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  • 5
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    Inactivation of the MSTN gene expression changes the composition and function of the gut microbiome in sheep
    Springer Science and Business Media LLC ; 2022
    In:  BMC Microbiology Vol. 22, No. 1 ( 2022-11-11)
    Details
    In: BMC Microbiology, Springer Science and Business Media LLC, Vol. 22, No. 1 ( 2022-11-11)
    Abstract: Myostatin ( MSTN ) negatively regulates the muscle growth in animals and MSTN deficient sheep have been widely reported previously. The goal of this study was to explore how MSTN inactivation influences their gut microbiota composition and potential functions. Results We compared the slaughter parameters and meat quality of 3 MSTN -edited male sheep and 3 wild-type male sheep, and analyzed the gut microbiome of the MSTN -edited sheep (8 female and 8 male sheep) and wild-type sheep (8 female and 8 male sheep) through metagenomic sequencing. The results showed that the body weight, carcass weight and eye muscle area of MSTN -edited sheep were significantly higher, but there were no significant differences in the meat quality indexes. At the microbial level, the alpha diversity was significantly higher in the MSTN -edited sheep ( P   〈  0.05), and the microbial composition was significantly different by PCoA analysis in the MSTN -edited and wild-type sheep. The abundance of Firmicutes significantly increased and Bacteroidota significantly decreased in the MSTN -edited sheep. At genus level, the abundance of Flavonifractor, Subdoligranulum, Ruthenibacterium, Agathobaculum, Anaerotignum, Oribacterium and Lactobacillus were significantly increased in the MSTN -edited sheep ( P   〈  0.05). Further analysis of functional differences was found that the carotenoid biosynthesis was significantly increased and the peroxisome, apoptosis, ferroptosis, N-glycan biosynthesis, thermogenesis, and adipocytokines pathways were decreased in the MSTN -edited sheep ( P   〈  0.05). Moreover, carbohydrate-active enzymes (CAZymes) results certified the abundance of the GH13_39, GH4, GH137, GH71 and PL17 were upregulated, and the GT41 and CBM20 were downregulated in the MSTN -edited sheep ( P   〈  0.05). Conclusions Our study suggested that MSTN inactivation remarkably influenced the composition and potential function of hindgut microbial communities of the sheep, and significantly promoted growth performance without affecting meat quality.
    Type of Medium: Online Resource
    ISSN: 1471-2180
    URL: Article
    DOI: 10.1186/s12866-022-02687-8
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 2041505-9
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  • 6
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    Comparative transcriptome analysis reveals potentially novel roles of Homeobox genes in adipose deposition in fat-tailed sheep
    Springer Science and Business Media LLC ; 2017
    In:  Scientific Reports Vol. 7, No. 1 ( 2017-11-03)
    Details
    In: Scientific Reports, Springer Science and Business Media LLC, Vol. 7, No. 1 ( 2017-11-03)
    Abstract: Adipose tissues are phenotypically, metabolically and functionally heterogeneous based on the sites of their deposition. Undesirable fat deposits in the body are often detrimental to animal and human health. To unravel the potential underlying mechanisms governing accumulation of adipose tissues in various regions of the body, i.e., subcutaneous (SAT), visceral (VAT) and tail (TAT), we profiled transcriptomes from Tan sheep, a Chinese indigenous breed with notable fat tail using RNA-seq. Upon comparison, we identified a total of 1,058 differentially expressed genes (DEGs) between the three adipose types (218, 324, and 795 in SAT/VAT, SAT/TAT, and VAT/TAT, respectively), from which several known key players were identified that are involved in lipid metabolic process, Wnt signals, Vitamin A metabolism, and transcriptional regulation of adipocyte differentiation. We also found that many elevated genes in VAT were notably enriched for key biological processes such as cytokine secretion, signaling molecule interaction and immune systems. Several developmental genes including HOXC11, HOXC12 and HOXC13, and adipose-expressed genes in the tail region, such as HOTAIR_2, HOTAIR_3 and SP9 were specially highlighted, indicating their strong associations with tail fat development in fat-tailed sheep. Our results provide new insight into exploring the specific fat deposition in tail, also contribute to the understanding of differences between adipose depots.
    Type of Medium: Online Resource
    ISSN: 2045-2322
    URL: Article
    DOI: 10.1038/s41598-017-14967-9
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2017
    detail.hit.zdb_id: 2615211-3
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  • 7
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    Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model
    CSIRO Publishing ; 2019
    In:  Reproduction, Fertility and Development Vol. 31, No. 9 ( 2019), p. 1533-
    Details
    In: Reproduction, Fertility and Development, CSIRO Publishing, Vol. 31, No. 9 ( 2019), p. 1533-
    Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9:sgRNA (sg8) and found that injection with 50ngμL−1 Cas9 mRNA and 25ngμL−1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.
    Type of Medium: Online Resource
    ISSN: 1031-3613
    URL: Article
    DOI: 10.1071/RD18485
    Language: English
    Publisher: CSIRO Publishing
    Publication Date: 2019
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  • 8
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    Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality
    Springer Science and Business Media LLC ; 2022
    In:  BMC Genomics Vol. 23, No. 1 ( 2022-12)
    Details
    In: BMC Genomics, Springer Science and Business Media LLC, Vol. 23, No. 1 ( 2022-12)
    Abstract: CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. Results We report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin ( MSTN ) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency 〉  50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN -knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN -edited lambs were also investigated. MSTN -knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN -knockout lambs did not show changes compared to the wild-type lambs. Conclusions This study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN -knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.
    Type of Medium: Online Resource
    ISSN: 1471-2164
    URL: Article
    DOI: 10.1186/s12864-022-08594-6
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 2041499-7
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  • 9
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    Genetics of the phenotypic evolution in sheep: a molecular look at diversity-driving genes
    Springer Science and Business Media LLC ; 2022
    In:  Genetics Selection Evolution Vol. 54, No. 1 ( 2022-09-09)
    Details
    In: Genetics Selection Evolution, Springer Science and Business Media LLC, Vol. 54, No. 1 ( 2022-09-09)
    Abstract: After domestication, the evolution of phenotypically-varied sheep breeds has generated rich biodiversity. This wide phenotypic variation arises as a result of hidden genomic changes that range from a single nucleotide to several thousands of nucleotides. Thus, it is of interest and significance to reveal and understand the genomic changes underlying the phenotypic variation of sheep breeds in order to drive selection towards economically important traits. Review Various traits contribute to the emergence of variation in sheep phenotypic characteristics, including coat color, horns, tail, wool, ears, udder, vertebrae, among others. The genes that determine most of these phenotypic traits have been investigated, which has generated knowledge regarding the genetic determinism of several agriculturally-relevant traits in sheep. In this review, we discuss the genomic knowledge that has emerged in the past few decades regarding the phenotypic traits in sheep, and our ultimate aim is to encourage its practical application in sheep breeding. In addition, in order to expand the current understanding of the sheep genome, we shed light on research gaps that require further investigation. Conclusions Although significant research efforts have been conducted in the past few decades, several aspects of the sheep genome remain unexplored. For the full utilization of the current knowledge of the sheep genome, a wide practical application is still required in order to boost sheep productive performance and contribute to the generation of improved sheep breeds. The accumulated knowledge on the sheep genome will help advance and strengthen sheep breeding programs to face future challenges in the sector, such as climate change, global human population growth, and the increasing demand for products of animal origin.
    Type of Medium: Online Resource
    ISSN: 1297-9686
    URL: Article
    DOI: 10.1186/s12711-022-00753-3
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2022
    detail.hit.zdb_id: 2012369-3
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  • 10
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    Data_Sheet_1.xls
    Frontiers Media SA ; 2018
    In:  Frontiers in Genetics Vol. 9 ( 2018-10-4)
    Details
    In: Frontiers in Genetics, Frontiers Media SA, Vol. 9 ( 2018-10-4)
    Type of Medium: Online Resource
    ISSN: 1664-8021
    URL: Article
    URL: Article
    URL: Article
    DOI: 10.3389/fgene.2018.00449
    DOI: 10.3389/fgene.2018.00449.s001
    DOI: 10.3389/fgene.2018.00449.s002
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2018
    detail.hit.zdb_id: 2606823-0
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