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Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12i^(Max)

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Science China(Life Sciences) 2024年第3期67卷 555-564页

作者： Jilong Ren Tang Hai Yangcan Chen Ke Sun Zhiqiang Han Jing Wang Chongyang Li Qingwei Wang Leyun Wang Huabing Zhu Dawei Yu Wei Li Shanjiang Zhao State Key Laboratory of animal Nutrition Key Laboratory of Animal GeneticsBreeding and Reproduction of Ministry of Agriculture and Rural AffairsInstitute of Animal ScienceChinese Academy of Agricultural SciencesBeijing 100193China State Key Laboratoryf Stem Cell and Reproductive Biology Instituteof ZooogyChinese Academyof SciencesBeijing 100101China Beijing institute for Stem Cell and Regenerative Medicine Beijing 100101China Beijing Farm animal research Center Institute of ZoologyChinese Academy of SciencesBeijing 100101China

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated gene(Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12i^(Max), a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12i^(Max)in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163,and MSTN via Cas12i^(Max)in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12i^(Max)for gene editing in livestock animals and demonstrated the potential application of Cas12i^(Max)in the field of animal trait improvement for agricultural production.

关键词： Cas12i^(Max) gene editing livestock multiple trait improvement

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Hepatic steatosis is associated with dysregulated cholesterol metabolism and altered protein acetylation dynamics in chickens

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Journal of animal Science and Biotechnology 2024年第1期15卷 53-67页

作者： Xiaoli Guo Qianqian Zhou Jiaming Jin Fangren Lan Chaoliang Wen Junying Li Ning Yang Congjiao Sun National Engineering Laboratory for animal Breeding and Key Laboratory of animal Genetics Breeding and ReproductionMinistry of Agriculture and Rural AffairsChina Agricultural UniversityBeijing 100193China

Background Hepatic steatosis is a prevalent manifestation of fatty liver, that has detrimental effect on the health and productivity of laying hens, resulting in economic losses to the poultry industry. Here, we aimed to systematically investigate the genetic regulatory mechanisms of hepatic steatosis in laying *** Ninety individuals with the most prominent characteristics were selected from 686 laying hens according to the accumulation of lipid droplets in the liver, and were graded into three groups, including the control, mild hepatic steatosis and severe hepatic steatosis groups. A combination of transcriptome, proteome, acetylome and lipidome analyses, along with bioinformatics analysis were used to screen the key biological processes, modifications and lipids associated with hepatic *** The rationality of the hepatic steatosis grouping was verified through liver biochemical assays and RNA-seq. Hepatic steatosis was characterized by increased lipid deposition and multiple metabolic abnormalities. Integration of proteome and acetylome revealed that differentially expressed proteins(DEPs) interacted with differentially acetylated proteins(DAPs) and were involved in maintaining the metabolic balance in the liver. Acetylation alterations mainly occurred in the progression from mild to severe hepatic steatosis, i.e., the enzymes in the fatty acid oxidation and bile acid synthesis pathways were significantly less acetylated in severe hepatic steatosis group than that in mild group(P < 0.05). Lipidomics detected a variety of sphingolipids(SPs) and glycerophospholipids(GPs) were negatively correlated with hepatic steatosis(r ≤-0.5, P < 0.05). Furthermore, the severity of hepatic steatosis was associated with a decrease in cholesterol and bile acid synthesis and an increase in exogenous cholesterol *** In addition to acquiring a global and thorough picture of hepatic steatosis in laying hens, we were able to reveal the role of ac

关键词： Acetylation Cholesterol metabolism Hepatic steatosis Laying hens Multiomics

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Biotic and abiotic stresses on honeybee health

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Integrative Zoology 2024年第3期19卷 442-457页

作者： Zheguang LIN Siyi SHEN Kang WANG Ting JI Apicultural research institute College of Animal Science and TechnologyYangzhou UniversityYangzhouChina

Honeybees are the most critical pollinators providing key ecosystem services that underpin crop production and sustainable *** a backdrop of rapid global change,this eusocial insect encounters a succession of stressors during nesting,foraging,and *** mites,together with vectored viruses,have been recognized as central biotic threats to honeybee health,while the spread of invasive giant hornets and small hive beetles also increasingly threatens colonies *** of agrochemicals,including acaricides used for mite treatment,and other pollutants of the environment have been widely documented to affect bee health in various ***,expanding urbanization,climate change,and agricultural intensification often result in the destruction or fragmentation of flower-rich bee *** anthropogenic pressures exerted by beekeeping management practices affect the natural selection and evolution of honeybees,and colony translocations facilitate alien species invasion and disease *** this review,the multiple biotic and abiotic threats and their interactions that potentially undermine bee colony health are discussed,while taking into consideration the sensitivity,large foraging area,dense network among related nestmates,and social behaviors of honeybees.

关键词： abiotic stressor Apis biotic stressor colony losses interaction effect pollinator decline

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Asymmetric expression of CA2 and CA13 linked to calcification in the bilateral mandibular condyles cause crossed beaks in chickens

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Journal of Integrative Agriculture 2024年第7期23卷 2379-2390页

作者： Lei Shi Yanyan Sun Yunlei Li Hao Bai Jingwei Yuan Hui Ma Yuanmei Wang Panlin Wang Aixin Ni Linlin Jiang Pingzhuang Ge Shixiong Bian Yunhe Zong Jinmeng Zhao Adamu MIsa Hailai HTesfay Jilan Chen State Key Laboratory of animal Biotech Breeding/Key Laboratory of animal(Poultry)Genetics Breeding and reproduction of Ministry of Agriculture and Rural Affairs/institute of animal Sciences Chinese Academy of Agricultural SciencesBeijing 100193China College of animal Science and Technology Hebei Agricultural UniversityBaoding 071001China Joint International research Laboratory of Agriculture and Agri-Product Safety Ministry of Education/Institutes of Agricultural Science and Technology DevelopmentYangzhou UniversityYangzhou 225009China

Crossed beak is a complex mode of inheritance with prevalence ranging from 0.2 to 7.4% in at least 12 chicken strains *** reveal the intrinsic factors causing crossed beaks,genes expression patterns in bilateral mandibular condyle between affected and normal birds were characterized by RNA sequencing analysis in the present *** beak was induced by short length of unilateral mandibular ramus,and a total of 110differentially expressed genes were up-or down-regulated in the affected(short)mandibular condyle side as compared to the normal *** anhydrase 2(CA2)and Carbonic anhydrase 13(CA13)were enriched in the carbonate dehydratase activity,and high-expressed in mandibular condyle and osteoblasts(Presearch suggested that CA2 and CA13 are down-calcified in shortside mandibular condyle,and caused mandibular ramus to grow ***2 and CA13 have the critical role in crossed beaks by regulating calcification of mandibular condyle.

关键词： chicken crossed beak carbonic anhydrase calcification CA2 CA13

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Transcriptome analysis reveals steroid hormones biosynthesis pathway involved in abdominal fat deposition in broilers

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Journal of Integrative Agriculture 2024年第9期 3118-3128页

作者： Yuting Zhu Yongli Wang Yidong Wang Guiping Zhao Jie Wen Huanxian Cui State Key Laboratory of animal Nutrition/Key Laboratory of animal (Poultry) Genetics Breeding and reproduction Ministry of Agriculture and Rural Affairs/Institute of Animal SciencesChinese Academy of Agricultural Sciences

Excessive abdominal fat deposition reduces the feed efficiency and increase the cost of production in broilers. Therefore, it is an important task for poultry breeders to breed broilers with low abdominal fat. Abdominal fat deposition is a highly complex biological process, and its molecular basis remains elusive. In this study, we performed transcriptome analysis to compare gene expression profiles at different stages of abdominal fat deposition to identify the key genes and pathways involved in abdominal fat accumulation. We found that abdominal fat weight(AFW) increased gradually from day 35(D35) to 91(D91), and then decreased at day 119(D119). Accordingly, after detecting differentially expressed genes(DEGs) by comparing gene expression profiles at D35 vs. D63 and D35 vs. D91, and identifying gene modules associated with fat deposition by weighted gene co-expression network analysis(WGCNA), we performed intersection analysis of the detected DEGs and WGCNA gene modules and identified 394 and 435 intersecting genes, respectively. The results of the Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses showed that the steroid hormone biosynthesis and insulin signaling pathways were co-enriched in all intersecting genes, steroid hormones have been shown that regulated insulin signaling pathway, indicating the importance of the steroid hormone biosynthesis pathway in the development of broiler abdominal fat. We then identified 6 hub genes(ACTB, SOX9, RHOBTB2, PDLIM3, NEDD9, and DOCK4) related to abdominal fat deposition. Further analysis also revealed that there were direct interactions between 6 hub genes. SOX9 has been shown to bind to proteins required for steroid hormone receptor binding, and RHOBTB2 indirectly regulates the steroid hormones biosynthesis through cyclin factor, and ultimately affect fat deposition. Our results suggest that the genes RHOBTB2 and SOX9 play an important role in

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3D genome organization and its study in livestock breeding

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Journal of Integrative Agriculture 2024年第1期23卷 39-58页

作者： Jie Cheng Xiukai Cao Shengxuan Wang Jiaqiang Zhang Binglin Yue Xiaoyan Zhang Yongzhen Huang Xianyong Lan Gang Ren Hong Chen Key Laboratory of animal Genetics Breeding and Reproduction of Shaanxi ProvinceCollege of Animal Science and TechnologyNorthwest A&F UniversityYangling 712100China Joint International research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China Yangzhou UniversityYangzhou 225009China College of animal Science Xinjiang Agricultural UniversityUrumqi 830052China

Eukaryotic genomes are hierarchically packaged into cell nucleus,affecting gene *** genome is organized into multiscale structural units,including chromosome territories,compartments,topologically associating domains(TADs),and DNA *** identification of these hierarchical structures has benefited from the development of experimental approaches,such as 3C-based methods(Hi-C,ChIA-PET,etc.),imaging tools(2D-FISH,3D-FISH,Cryo-FISH,etc.)and ligation-free methods(GAM,SPRITE,etc.).In recent two decades,numerous studies have shown that the 3D organization of genome plays essential roles in multiple cellular processes via various mechanisms,such as regulating enhancer activity and promoter-enhancer ***,there are relatively few studies about the 3D genome in livestock ***,studies for exploring the function of 3D genomes in livestock are urgently needed to provide a more comprehensive understanding of potential relationships between the genome and production *** this review,we summarize the recent advances of 3D genomics and its biological functions in human and mouse studies,drawing inspiration to explore the 3D genomics of livestock *** then mainly focus on the biological functions of 3D genome organization in muscle development and its implications in animal breeding.

关键词： 3D genome organization 3D genomic methodology regulatory mechanisms muscle development livestock breeding

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Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction

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Journal of animal Science and Biotechnology 2024年第1期15卷 171-181页

作者： Estel Viñolas-Vergés Jordi Ribas-Maynou Isabel Barranco Camila Peres Rubio Sergi Bonet Jordi Roca Marc Yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGironaSpain Biotechnology of animal and Human reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGironaSpain Department of Medicine and animal Surgery Faculty of Veterinary ScienceUniversity of MurciaMurciaSpain Catalan Institution for research and Advanced Studies(ICREA) S08010 BarcelonaSpain

Background Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo *** some mammals,like pigs,ejaculates are emitted in three separate fractions:pre-sperm,sperm-rich(SRF)and post sperm-rich(PSRF).These fractions are known to vary in volume,sperm concentration and quality,as well as in the origin and composition of seminal plasma(SP),with differences being also observed within the SRF ***,whether disparities in the DNA integrity and chromatin condensation and pro-tamination of their sperm exist has not been *** This study determined chromatin protamination(Chromomycin A3 test,CMA_(3)),condensation(Dibromobi-mane test,DBB),and DNA integrity(Comet assay)in the pig sperm contained in the first 10 m L of the SRF(SRF-P1),the remaining portion of the sperm-rich fraction(SRF-P2),and the post sperm-rich fraction(PSRF).While chromatin protamination was found to be similar between the different ejaculate fractions(P>0.05),chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF(P=0.018 and P=0.004,respectively).Regarding DNA integrity,no differences between fractions were observed(P>0.05).As the SRF-P1 has the highest sperm concentra-tion and ejaculate fractions are known to differ in antioxidant composition,the oxidative stress index(OSi)in SP,calcu-lated as total oxidant activity divided by total antioxidant capacity,was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF(0.42±0.06 vs.0.23±0.09 and 0.08±0.00,respectively;P<0.01);this index,in addition,was observed to be correlated to the sperm concentration of each fraction(Rs=0.973;P<0.001).Conclusion While sperm DNA integrity was not found to differ between ejaculate fractions,SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the *** could be related to the OSi of each fraction.

关键词： Chromatin Condensation DNA integrity Ejaculate fractions Pig Protamination Sperm

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Dynamic transcriptome profiles and novel markers in bovine spermatogenesis revealed by single-cell sequencing

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Journal of Integrative Agriculture 2024年第7期23卷 2362-2378页

作者： Yuan Gao Fuxia Bai Qi Zhang Xiaoya An Zhaofei Wang Chuzhao Lei Ruihua Dang Key Laboratory of animal Genetics Breeding and Reproduction of Shaanxi ProvinceCollege of Animal Science and TechnologyNorthwest A&F UniversityYangling 712100China College of Life Sciences Northwestern Polytechnical UniversityXi’an 710072China

Testicular development is an important biological process in male and requires interaction between the male germ cells and somatic ***,the mechanisms of testicular development in livestock,particularly in cattle,are poorly ***,cellular heterogeneity hinders the profiling of different cell types at different developmental *** this study,we first performed a single-cell transcriptomic study of the bovine testis development during puberty by using 10×genomics single-cell RNA sequencing(scRNA-seq).By collecting the scRNA-seq data from 11,083 cells from prepubertal and pubertal bovine testes,a high-resolution scRNA-seq atlas was described,identifying 9 somatic and 13 spermatogenic *** also distinguished several stage-specific marker genes for bovine germ cells and somatic cells,such as GRAF2 and MORC1 for SSC(spermatogonial stem cells),HJURP and TCF19 for differentiating spermatogonia,ARSE for immature Sertoli,CLEC12B for mature Sertoli,LOC112441470 for *** conclusion,we have examined the transcription levels and constructed the single-cell developmental maps of germ cells and somatic cells during testicular development in Angus *** datasets provided new insights into spermatogenesis and testicular somatic cell development in cattle.

关键词： scRNA-seq cattle spermatogenesis testis development

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Hexokinase 1 and 2 mediates glucose utilization to regulate the synthesis of kappa casein via ribosome protein subunit 6 kinase 1 in bovine mammary epithelial cells

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animal Nutrition 2024年第1期16卷 338-349页

作者： Tianyu Yang Jia Guo Han Song Osmond Datsomor Yuhang Chen Maocheng Jiang Kang Zhan Guoqi Zhao institute of animal Culture Collection and Application College of Animal Science and Technology Yangzhou University

Glucose plays a vital part in milk protein synthesis through the mTOR signaling pathway in bovine mammary epithelial cells(BMEC). The objectives of this study were to determine how glucose affects hexokinase(HK) activity in BMEC and investigate the regulatory effect of HK in kappa casein(CSN3)synthesis via the mechanistic target of rapamycin complex 1(mTORC1) signaling pathway in BMEC. For this, HK1 and HK2 were knocked out in BMEC using the CRISPR/Cas9 system. The gene and protein expression, glucose uptake, and cell proliferation were measured. We found that glucose uptake, cell proliferation, CSN3 gene expression levels, and expression of HK1 and HK2 increased with increasing glucose concentrations. Notably, glucose uptake was significantly reduced in HK2 knockout(HK2KO)BMEC treated with 17.5 mM glucose. Moreover, under the same glucose treatment conditions, the proliferative ability and abundance of CSN3 were significantly diminished in both HK1 knockout(HK1KO)and HK2KO BMEC compared with that in wild-type BEMC. We further observed that the phosphorylation levels of ribosome protein subunit 6 kinase 1(S6K1) were reduced in HK1KO and HK2KO BMEC following treatment with 17.5 mM glucose. As expected, the levels of glucose-6-phosphate and the m RNA expression levels of glycolysis-related genes were decreased in both HK1KO and HK2KO BMEC following glucose treatment. These results indicated that the knockout of HK1 and HK2 inhibited cell proliferation and CSN3 expression in BMEC under glucose treatment, which may be associated with the inactivation of the S6K1 and inhibition of glycolysis.

关键词： Glucose Hexokinase Milk protein Mechanistic target of rapamycin complex 1 signaling pathway Bovine mammary epithelial cell Kappa casein

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Sperm function, mitochondrial activity and in vivo fertility are associated to their mitochondrial DNA content in pigs

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Journal of animal Science and Biotechnology 2024年第4期15卷 1550-1558页

作者： Marc Llavanera Yentel Mateo‑Otero Estel Vinolas‑Vergés Sergi Bonet Marc Yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGirona ES‑17003Spain Biotechnology of animal and Human reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGirona ES‑17003Spain Catalan Institution for research and Advanced Studies(ICREA) Barcelona ES‑08010Spain

Background Despite their low abundance in sperm, mitochondria have diverse functions in this cell type, includ-ing energy production, signalling and calcium regulation. In humans, sperm mitochondrial DNA content(mtDNAc) has been reported to be negatively linked to sperm function and fertility. Yet, the association between mtDNAc and sperm function in livestock remains unexplored. For this reason, this study aimed to shed some light on the link between mtDNAc and sperm function and fertilising potential in pigs. A qPCR method for mtDNAc quantification was optimised for pig sperm, and the association of this parameter with sperm motility, kinematics, mitochondrial activity, and fertility was subsequently *** First, the q PCR method was found to be sensitive and efficient for mtDNAc quantification in pig sperm. By using this technique, mtDNAc was observed to be associated to sperm motility, mitochondrial activity and in vivo, but not in vitro, fertility outcomes. Specifically, sperm with low mtDNAc were seen to exhibit greater motility but decreased mitochondrial activity and intracellular reactive oxygen species. Interestingly, samples with lower mtD-NAc showed higher conception and farrowing rates, but similar in vitro fertilisation rates and embryo development, when compared to those with greater *** These findings enrich our comprehension of the association of mtDNAc with sperm biology, and lay the foundation for future research into employing this parameter as a molecular predictor for sperm function and fer-tility in livestock.

关键词： Fertility Mitochondria Motility mtDNA copy number qPCR Sperm

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