Guangxian Zhou

Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system

Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding.

Transcriptome profile analysis of adipose tissues from fat and short-tailed sheep

Recent studies in domestic animals have used RNA-seq to explore the transcriptome of different tissues in a limited number of individuals. In the present study, de novo transcriptome sequencing was used to compare sheep adipose tissue transcriptome profiles between a fat-tailed breed (Kazak sheep; KS) and a short-tailed (Tibetan sheep; TS). The RNA-seq data from these two groups revealed that 646 genes were differentially expressed between the KS and TS groups, including 280 up-regulated and 366 down-regulated genes. We identified genes relevant to fat metabolism in adipose tissues, including two top genes with the largest fold change (NELL1 and FMO3). Pathway analysis revealed that the differentially expressed genes between the KS and TS breeds belong to fatty acid metabolism relevant pathways (e.g. fat digestion and absorption, glycine, serine, and threonine metabolism) and cell junction-related pathways (e.g. cell adhesion molecules) which contribute to fat deposition. This work highlighted potential genes and gene networks that affect fat deposition and meat quality in sheep.

Novel polymorphisms of the growth hormone gene and their effect on growth traits in Chinese goats

The polymorphisms of the growth hormone (GH) gene were analyzed in 686 individuals from four goat populations, Three haplotypes (A, B and C) and three observed genotypes (AA, AB and AC) were detected at the P2 locus, and three haplotypes (E, F and G) and three observed genotypes (EE, EF and EG) were also detected at the P4 locus. In addition, five single nucleotide polymorphisms (SNPs)-A112G, C142T (Gly>Ser), C214T (P2 locus), C266A (Pro>His) and C214T (P4 locus, Arg>Trp), were identified by GH gene sequencing and PCR-SSCP analysis. The SNPs loci were in Hardy-Weinberg disequilibrium in three goat populations (P<0.05). Association of polymorphisms with growth traits was done in BG, F1 and F1 populations, which were shown to be associated with growth traits in three goat populations. The SNPs in the goat GH gene had significant effects on growth traits (P<0.05). suggesting that the GH gene is a strong candidate gene that affects growth traits in goat.

Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep

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.

Whole-genome sequencing of eight goat populations for the detection of selection signatures underlying production and adaptive traits.

The goat (Capra hircus) is one of the first farm animals that have undergone domestication and extensive natural and artificial selection by adapting to various environments, which in turn has resulted in its high level of phenotypic diversity. Here, we generated medium-coverage (9–13×) sequences from eight domesticated goat breeds, representing morphologically or geographically specific populations, to identify genomic regions representing selection signatures. We discovered ~10 million single nucleotide polymorphisms (SNPs) for each breed. By combining two approaches, ZHp and di values, we identified 22 genomic regions that may have contributed to the phenotypes in coat color patterns, body size, cashmere traits, as well as high altitude adaptation in goat populations. Candidate genes underlying strong selection signatures including coloration (ASIP, KITLG, HTT, GNA11, and OSTM1), body size (TBX15, DGCR8, CDC25A, and RDH16), cashmere traits (LHX2, FGF9, and WNT2), and hypoxia adaptation (CDK2, SOCS2, NOXA1, and ENPEP) were identified. We also identified candidate functional SNPs within selected genes that may be important for each trait. Our results demonstrated the potential of using sequence data in identifying genomic regions that are responsible for agriculturally significant phenotypes in goats, which in turn can be used in the selection of goat breeds for environmental adaptation and domestication.

Comparative Transcriptome Analysis of Fetal Skin Reveals Key Genes Related to Hair Follicle Morphogenesis in Cashmere Goats.

Cashmere goat skin contains two types of hair follicles (HF): primary hair follicles (PHF) and secondary hair follicles (SHF). Although multiple genetic determinants associated with HF formation have been identified, the molecules that determine the independent morphogenesis of HF in cashmere goats remain elusive. The growth and development of SHF directly influence the quantity and quality of cashmere production. Here, we report the transcriptome profiling analysis of nine skin samples from cashmere goats using 60- and 120-day-old embryos (E60 and E120, respectively), as well as newborns (NB), through RNA-sequencing (RNA-seq). HF morphological changes indicated that PHF were initiated at E60, with maturation from E120, while differentiation of SHF was identified at E120 until formation of cashmere occurred after birth (NB). The RNA-sequencing analysis generated over 20.6 million clean reads from each mRNA library. The number of differentially expressed genes (DEGs) in E60 vs. E120, E120 vs. NB, and E60 vs. NB were 1,024, 0 and 1,801, respectively, indicating that no significant differences were found at transcriptomic levels between E120 and NB. Key genes including B4GALT4, TNC, a-integrin, and FGFR1, were up-regulated and expressed in HF initiation from E60 to E120, while regulatory genes such as GPRC5D, PAD3, HOXC13, PRR9, VSIG8, LRRC15, LHX2, MSX-2, and FOXN1 were up-regulated and expressed in HF keratinisation and hair shaft differentiation from E120 and NB to E60. Several genes belonging to the KRT and KRTAP gene families were detected throughout the three HF developmental stages. The transcriptional trajectory analyses of all DEGs indicated that immune privilege, glycosaminoglycan biosynthesis, extracellular matrix receptor interaction, and growth factor receptors all played dominant roles in the epithelial-mesenchymal interface and HF formation. We found that the Wnt, transforming growth factor-beta/bone morphogenetic protein, and Notch family members played vital roles in HF differentiation and maturation. The DEGs we found could be attributed to the generation and development of HF, and thus will be critically important for improving the quantity and quality of fleece production in animals for fibres.

Fibroblast growth factor 5-short (FGF5s) inhibits the activity of FGF5 in primary and secondary hair follicle dermal papilla cells of cashmere goats.

To determine the relationship between fibroblast growth factor 5 (FGF5) and FGF5-short (FGF5s) in dermal papilla cells of cashmere goat primary and secondary hair follicles. We isolated dermal papilla cells from primary hair follicle (PHF) and secondary hair follicle (SHF) of cashmere goat, and found that the FGF5 receptor, fibroblast growth factor receptor 1 (FGFR1), was expressed in these two types of dermal papilla cells. Moreover, adenovirus-mediated overexpression of FGF5 could upregulate the mRNA expression of insulin-like growth factor-1 (IGF-1), versican and noggin that were important for follicle growth maintenance, whereas downregulate the expression of anagen chalone bone morphogenetic protein 4 (BMP4) in dermal papilla cells. However, these alterations were partly reversed by FGF5s overexpression. In conclusion, our results demonstrated that FGF5s acted as an inhibitor of FGF5 in the regulation of anagen-catagen transition of cashmere goat dermal papilla cells.

Integrating miRNA and mRNA Expression Profiling Uncovers miRNAs Underlying Fat Deposition in Sheep

MicroRNAs (miRNAs) are endogenous, noncoding RNAs that regulate various biological processes including adipogenesis and fat metabolism. Here, we adopted a deep sequencing approach to determine the identity and abundance of miRNAs involved in fat deposition in adipose tissues from fat-tailed (Kazakhstan sheep, KS) and thin-tailed (Tibetan sheep, TS) sheep breeds. By comparing HiSeq data of these two breeds, 539 miRNAs were shared in both breeds, whereas 179 and 97 miRNAs were uniquely expressed in KS and TS, respectively. We also identified 35 miRNAs that are considered to be putative novel miRNAs. The integration of miRNA-mRNA analysis revealed that miRNA-associated targets were mainly involved in the gene ontology (GO) biological processes concerning cellular process and metabolic process, and miRNAs play critical roles in fat deposition through their ability to regulate fundamental pathways. These pathways included the MAPK signaling pathway, FoxO and Wnt signaling pathway, and focal adhesion. Taken together, our results define miRNA expression signatures that may contribute to fat deposition and lipid metabolism in sheep.

Comparative proteomic analyses using iTRAQ-labeling provides insights into fiber diversity in sheep and goats

The structural component of wool and hair fibers, such as keratin-associated proteins (KAPs), has been well described, but the genetic determinants of fiber diameter are largely unknown. Here, we have used an iTRAQ-based proteomic approach to investigate differences in protein abundance among 18 samples from sheep and goats across a diverse range of fibers. We identified proteins with different abundance and are associated with variation in fiber features. Proteins with different abundance are mainly keratin or keratin-associated proteins (KRTAP11-1, KRT6A, KRT38), or are related to hair growth (DSC2, DSG3, EEF2, CALML5, TCHH, SELENBP1) and fatty acid synthesis (FABP4, FABP5). RNA-seq further confirmed the functional importance of the DSC2 gene in the determination of woolly phenotype in goat fibers. This comprehensive analysis of fibers from major fiber-producing animals is the first to provide a list of candidate proteins that are involved in fiber formation. This list will be valuable asset for future studies into the molecular mechanisms that underlie fiber diversity. BIOLOGICAL SIGNIFICANCE Proteins are the basis for animal-derived hair fibers, yet proteins conferring fiber structure and characteristics in sheep and goats are largely elusive. By examining 27 fibers samples representing 9 fiber types from sheep and goats through the iTRAQ approach, we show a list of differentially abundant proteins that are important to hair structural component, or genes related to hair growth and fatty acid synthesis. RNA-seq further validated the DSC2 gene is key to the woolly/straight hair phenotype in goats.

Integrative analysis reveals ncRNA-mediated molecular regulatory network driving secondary hair follicle regression in cashmere goats

BackgroundCashmere is a keratinized product derived from the secondary hair follicles (SHFs) of cashmere goat skins. The cashmere fiber stops growing following the transition from the actively proliferating anagen stage to the apoptosis-driven catagen stage. However, little is known regarding the molecular mechanisms responsible for the occurrence of apoptosis in SHFs, especially as pertains to the role of non-coding RNAs (ncRNAs) and their interactions with other molecules. Hair follicle (HF) degeneration is caused by localized apoptosis in the skin, while anti-apoptosis pathways may coexist in adjacent HFs. Thus, elucidating the molecular interactions responsible for apoptosis and anti-apoptosis in the skin will provide insights into HF regression.ResultsWe used multiple-omics approaches to systematically identify long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs expressed in cashmere goat skins in two crucial phases (catagen vs. anagen) of HF growth. Skin samples were collected from three cashmere goats at the anagen (September) and catagen (February) stages, and six lncRNA libraries and six miRNA libraries were constructed for further analysis. We identified 1122 known and 403 novel lncRNAs in the goat skins, 173 of which were differentially expressed between the anagen and catagen stages. We further identified 3500 gene-encoding transcripts that were differentially expressed between these two phases. We also identified 411 known miRNAs and 307 novel miRNAs, including 72 differentially expressed miRNAs. We further investigated the target genes of lncRNAs via both cis- and trans-regulation during HF growth. Our data suggest that lncRNAs and miRNAs act synergistically in the HF growth transition, and the catagen inducer factors (TGFβ1 and BDNF) were regulated by miR-873 and lnc108635596 in the lncRNA-miRNA-mRNA networks.ConclusionThis study enriches the repertoire of ncRNAs in goats and other mammals, and contributes to a better understanding of the molecular mechanisms of ncRNAs involved in the regulation of HF growth and regression in goats and other hair-producing species.