Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them ...Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.展开更多
In recent decades,gene-editing technologies,typically based on deoxyribonucleases to specifically modify genomic sequences,have dramatically remodeled various aspects of life sciences,including fundamental research,br...In recent decades,gene-editing technologies,typically based on deoxyribonucleases to specifically modify genomic sequences,have dramatically remodeled various aspects of life sciences,including fundamental research,breeding,and medical therapeutics.So far,four types of endonucleases have been adopted and optimized as gene-editing tools:meganuclease,ZFN,TALEN,and Cas nuclease from the CRISPR-Cas system.Each tool comes with its own advantages and limitations.Over the last ten years,RNA-guided Cas nucleases have been extensively investigated and successfully implemented in almost all mammalian cells due to their remarkable editing efficacy,high specificity,and flexibility in targeting the specific locus.Diverse Cas nuclease,together with meganuclease,ZFN,and TALEN,represent the key strategies for nuclease-based gene editing.However,systematic introductions and comparisons among four types of nucleases are not yet available.Here,we overview the capabilities of four types of nucleases along the development history of gene editing and describe the molecular mechanisms of substrate recognition and cleavage.Particularly,we summarize the promising CRISPR-Cas systems as well as modified tools applied for gene editing in the eukaryotic genome.Moreover,how the re-modulated nucleases and other nucleases,either naturally occurring or AI-designed,might manipulate DNA sequences is discussed and proposed.展开更多
The control of protein functions with light is valuable for spatiotemporal probing of biological systems.Current small-molecule photo-modulation methods include the light-induced uncaging of inhibitors and chromophore...The control of protein functions with light is valuable for spatiotemporal probing of biological systems.Current small-molecule photo-modulation methods include the light-induced uncaging of inhibitors and chromophore-assisted light inactivation with reactive oxygen species(ROS).However,the constant target protein expression results in inadequate photo-modulation efficiency,particularly for less potent inhibitors and chromophores.Herein,we report a novel bifunctional small-molecule ligands strategy to photo-modulate gene-editing enzymes CRISPR/Cas9.A coumarin-derived small-molecule ligand Bhc-BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity.Our results highlight the synergistic photo-modulation with bifunctional small-molecule ligands,which offers a valuable addition to current CRISPR/Cas9 photo-modulation technologies and may extend to other protein classes.展开更多
CRISPR is an adaptive immune defense system found in bacteria and archaea that is resistant to heterologous invasive genetic material.Later studies showed that the CRISPR system can be used for gene editing.This study...CRISPR is an adaptive immune defense system found in bacteria and archaea that is resistant to heterologous invasive genetic material.Later studies showed that the CRISPR system can be used for gene editing.This study used the Web of Science database as a search object,then visually analyzed the literature related to CRISPR gene-editing technology with CiteSpaceV.The results show that publications had increased year by year.USA ranked first in terms of publications,China is second,but the centrality is very low.Doudna JA and Zhang F have made outstanding contributions.There are close connections between the internal institutions of the various states,but there are few links between the states.The hot spot and frontier are the application of CRISPR in animals,plants,detection,diagnosis,and clinical treatment.展开更多
Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a lar...Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a large genome,contain multiple nonessential regions that do not affect viral replication and are capable of accepting the insertion of an exogenous gene and expressing the antigen protein.Furthermore,animal alphaherpesviruses have a wide host spectrum,can replicate in the host and continuously stimulate the animal to produce immunity to the corresponding pathogen,thus making them ideal carriers for recombinant virus-vectored vaccines.With the development of gene-editing technology,recombinant viruses capable of expressing foreign genes can be constructed by various methods.Currently,studies on recombinant virusvectored vaccines constructed based on animal alphaherpesviruses have involved poultry,pigs,cattle,sheep,and companion animals.Studies have shown that the construction of recombinant animal alphaherpesviruses enables the acquisition of immunity to multiple diseases.This article mainly summarizes the current progress on animal alphaherpesvirus-vectored vaccines,aiming to provide reference for the development of new animal alphaherpesvirus-vectored vaccines.展开更多
Background:Experimental animals are used to study physiological phenomena,pathological mechanisms,and disease prevention.The gut microbiome is known as a potential confounding factor for inconsistent data from preclin...Background:Experimental animals are used to study physiological phenomena,pathological mechanisms,and disease prevention.The gut microbiome is known as a potential confounding factor for inconsistent data from preclinical studies.Although many gut microbiome studies have been conducted in recent decades,few have focused on gut microbiota fluctuation among representative mouse strains.Methods:A range of frequently used mouse strains were selected from 34 isolation packages representing disease-related animal(DRA),immunity defect animal(IDA),or gene-editing animal(GEA)from the BALB/c and C57BL/6J backgrounds together with normal mice,and their microbial genomic DNA were isolated from mouse feces to sequence for the exploration of gut microbiota.Results:Mouse background strain,classification,introduced source,introduced year,and reproduction type significantly affected the gut microbiota structure(p<0.001 for all parameters),with background strain contributing the greatest influence(R^(2)=0.237).In normal groups,distinct gut microbiota types existed in different mouse strains.Sixty-four core operational taxonomic units were obtained from normal mice,and 12 belonged to Lactobacillus.Interestingly,the gut microbiota in C57BL/6J was more stable than that in BALB/c mice.Furthermore,the gut microbiota in the IDA,GEA,and DRA groups significantly differed from that in normal groups(p<0.001 for all).Compared with the normal group,there was a significantly higher Chao 1 and Shannon index(p<0.001 for all)in the IDA,GEA,and DRA groups.Markedly changed classes occurred with Firmicutes and Bacteroidetes.The abundances of Helicobacter,Blautia,Enterobacter,Bacillus,Clostridioides,Paenibacillus,and Clostridiales all significantly decreased in the IDA,GEA,and DRA groups,whereas those of Saccharimonas,Rikenella,and Odoribacter all significantly increased.展开更多
Animal models of human diseases play a critical role in medical research.Pigs are anatomically and physiologically more like humans than are small rodents such as mice,making pigs an attractive option for modeling hum...Animal models of human diseases play a critical role in medical research.Pigs are anatomically and physiologically more like humans than are small rodents such as mice,making pigs an attractive option for modeling human diseases.Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease.In the present review,we summarize the current status of pig models for human cardiovascular,metabolic,neurodegenerative,and various genetic diseases.We also discuss areas that need to be improved.Animal models of human diseases play a critical role in medical research.Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease.In the present review,we summarize the current status of pig models for human cardiovascular,metabolic,neurodegenerative,various genetic diseases and xenotransplantation.展开更多
With supports from the National Natural Science Foundation of China and the National Key Research and Development Program,the research team led by Profs.Gao CaiXia(高彩霞)and Li JiaYang(李家洋)at the Institute of Gene...With supports from the National Natural Science Foundation of China and the National Key Research and Development Program,the research team led by Profs.Gao CaiXia(高彩霞)and Li JiaYang(李家洋)at the Institute of Genetics and Developmental Biology of Chinese Academy of Sciences and Associate Prof.Jiang LinJian(姜临建)at China Agricultural University,generated several herbicide-tolerant wheat germplasms using base editing to facilitate weed control in wheat fields.The paper,entitled“Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing,”was published in Nature Plants(doi:10.1038/s41477-019-0405-0).展开更多
Background:T-cell acute lymphoblastic leukemia(T-ALL)is an uncommon and aggressive subtype of acute lymphoblastic leukemia(ALL).In the serum of T-ALL patients,the activity of lactate dehydrogenase A(LDHA)is increased....Background:T-cell acute lymphoblastic leukemia(T-ALL)is an uncommon and aggressive subtype of acute lymphoblastic leukemia(ALL).In the serum of T-ALL patients,the activity of lactate dehydrogenase A(LDHA)is increased.We proposed that targeting LDHA may be a potential strategy to improve T-ALL outcomes.The current study was conducted to investigate the antileukemic effect of LDHA gene-targeting treatment on T-ALL and the underlying molecular mechanism.Methods:Primary T-ALL cell lines Jurkat and DU528 were treated with the LDH inhibitor oxamate.MTT,colony formation,apoptosis,and cell cycle assays were performed to investigate the effects of oxamate on T-ALL cells.Quantitative real-time PCR(qPCR)and Western blotting analyses were applied to determine the related signaling pathways.A mitochondrial reactive oxygen species(ROS)assay was performed to evaluate ROS production after T-ALL cells were treated with oxamate.A T-ALL transgenic zebrafish model with LDHA gene knockdown was established using CRISPR/Cas9 gene-editing technology,and then TUNEL,Western blotting,and T-ALL tumor progression analyses were conducted to investigate the effects of LDHA gene knockdown on T-ALL transgenic zebrafish.Results:Oxamate significantly inhibited proliferation and induced apoptosis of Jurkat and DU528 cells.It also arrested Jurkat and DU528 cells in G0/G1 phase and stimulated ROS production(all P<0.001).Blocking LDHA significantly decreased the gene and protein expression of c-Myc,as well as the levels of phosphorylated serine/threonine kinase(AKT)and glycogen synthase kinase 3 beta(GSK-3β)in the phosphatidylinositol 3′-kinase(PI3K)signaling pathway.LDHA gene knockdown delayed disease progression and down-regulated c-Myc mRNA and protein expression in T-ALL transgenic zebrafish.Conclusion:Targeting LDHA exerted an antileukemic effect on T-ALL,representing a potential strategy for T-ALL treatment.展开更多
基金the National Institutes of Health R21OD019934(KL)and U42OD011140(RSP)
文摘Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.
基金supported by the Ministry of Agriculture and Rural Affairs of Chinathe National Natural Science Foundation of China(32150018)start-up funds from Tsinghua University,Beijing(J.J.G.L.)
文摘In recent decades,gene-editing technologies,typically based on deoxyribonucleases to specifically modify genomic sequences,have dramatically remodeled various aspects of life sciences,including fundamental research,breeding,and medical therapeutics.So far,four types of endonucleases have been adopted and optimized as gene-editing tools:meganuclease,ZFN,TALEN,and Cas nuclease from the CRISPR-Cas system.Each tool comes with its own advantages and limitations.Over the last ten years,RNA-guided Cas nucleases have been extensively investigated and successfully implemented in almost all mammalian cells due to their remarkable editing efficacy,high specificity,and flexibility in targeting the specific locus.Diverse Cas nuclease,together with meganuclease,ZFN,and TALEN,represent the key strategies for nuclease-based gene editing.However,systematic introductions and comparisons among four types of nucleases are not yet available.Here,we overview the capabilities of four types of nucleases along the development history of gene editing and describe the molecular mechanisms of substrate recognition and cleavage.Particularly,we summarize the promising CRISPR-Cas systems as well as modified tools applied for gene editing in the eukaryotic genome.Moreover,how the re-modulated nucleases and other nucleases,either naturally occurring or AI-designed,might manipulate DNA sequences is discussed and proposed.
基金supported by the National Natural Science Foundation of China(22337005,22277133,91753126)the Youth Innovation Promotion Association(CAS 2023266)+1 种基金the CAS Interdisciplinary Innovation Team(JCTD-2020-16)the Program of Shanghai Academic/Technology Research Leader(21XD1424700).
文摘The control of protein functions with light is valuable for spatiotemporal probing of biological systems.Current small-molecule photo-modulation methods include the light-induced uncaging of inhibitors and chromophore-assisted light inactivation with reactive oxygen species(ROS).However,the constant target protein expression results in inadequate photo-modulation efficiency,particularly for less potent inhibitors and chromophores.Herein,we report a novel bifunctional small-molecule ligands strategy to photo-modulate gene-editing enzymes CRISPR/Cas9.A coumarin-derived small-molecule ligand Bhc-BRD0539 is developed to uncage the active inhibitor upon light irradiation and to generate ROS in the Cas9 proximity for the dual inhibition of Cas9 activity.Our results highlight the synergistic photo-modulation with bifunctional small-molecule ligands,which offers a valuable addition to current CRISPR/Cas9 photo-modulation technologies and may extend to other protein classes.
文摘CRISPR is an adaptive immune defense system found in bacteria and archaea that is resistant to heterologous invasive genetic material.Later studies showed that the CRISPR system can be used for gene editing.This study used the Web of Science database as a search object,then visually analyzed the literature related to CRISPR gene-editing technology with CiteSpaceV.The results show that publications had increased year by year.USA ranked first in terms of publications,China is second,but the centrality is very low.Doudna JA and Zhang F have made outstanding contributions.There are close connections between the internal institutions of the various states,but there are few links between the states.The hot spot and frontier are the application of CRISPR in animals,plants,detection,diagnosis,and clinical treatment.
基金supported by grants from the National Key Research and Development Program of China(2017YFD0500800)the earmarked fund for China Agriculture Research System(CARS-42-17)+1 种基金the Integration and Demonstration of Key Technologies for Goose Industrial Chain in Sichuan Province,China(2018NZ0005)the Sichuan Veterinary Medicine and Drug Innovation Group of China Agriculture Research System(SCCXTD-2020-18)。
文摘Recombinant virus-vectored vaccines are novel agents that can effectively activate specific and nonspecific immunity,are multivalent and multieffective,and have high safety ratings.Animal alphaherpesviruses have a large genome,contain multiple nonessential regions that do not affect viral replication and are capable of accepting the insertion of an exogenous gene and expressing the antigen protein.Furthermore,animal alphaherpesviruses have a wide host spectrum,can replicate in the host and continuously stimulate the animal to produce immunity to the corresponding pathogen,thus making them ideal carriers for recombinant virus-vectored vaccines.With the development of gene-editing technology,recombinant viruses capable of expressing foreign genes can be constructed by various methods.Currently,studies on recombinant virusvectored vaccines constructed based on animal alphaherpesviruses have involved poultry,pigs,cattle,sheep,and companion animals.Studies have shown that the construction of recombinant animal alphaherpesviruses enables the acquisition of immunity to multiple diseases.This article mainly summarizes the current progress on animal alphaherpesvirus-vectored vaccines,aiming to provide reference for the development of new animal alphaherpesvirus-vectored vaccines.
基金National Key R&D Program of ChinaGrant/Award Number:2021YFF0703400+3 种基金the National Natural Science Foundation of ChinaGrant/Award Number:General Program,82070103CAMS Innovation Fund for Medical ScienceGrant/Award Number:CIFMS,2021-I2M-1-036,2021-I 2M-1-034。
文摘Background:Experimental animals are used to study physiological phenomena,pathological mechanisms,and disease prevention.The gut microbiome is known as a potential confounding factor for inconsistent data from preclinical studies.Although many gut microbiome studies have been conducted in recent decades,few have focused on gut microbiota fluctuation among representative mouse strains.Methods:A range of frequently used mouse strains were selected from 34 isolation packages representing disease-related animal(DRA),immunity defect animal(IDA),or gene-editing animal(GEA)from the BALB/c and C57BL/6J backgrounds together with normal mice,and their microbial genomic DNA were isolated from mouse feces to sequence for the exploration of gut microbiota.Results:Mouse background strain,classification,introduced source,introduced year,and reproduction type significantly affected the gut microbiota structure(p<0.001 for all parameters),with background strain contributing the greatest influence(R^(2)=0.237).In normal groups,distinct gut microbiota types existed in different mouse strains.Sixty-four core operational taxonomic units were obtained from normal mice,and 12 belonged to Lactobacillus.Interestingly,the gut microbiota in C57BL/6J was more stable than that in BALB/c mice.Furthermore,the gut microbiota in the IDA,GEA,and DRA groups significantly differed from that in normal groups(p<0.001 for all).Compared with the normal group,there was a significantly higher Chao 1 and Shannon index(p<0.001 for all)in the IDA,GEA,and DRA groups.Markedly changed classes occurred with Firmicutes and Bacteroidetes.The abundances of Helicobacter,Blautia,Enterobacter,Bacillus,Clostridioides,Paenibacillus,and Clostridiales all significantly decreased in the IDA,GEA,and DRA groups,whereas those of Saccharimonas,Rikenella,and Odoribacter all significantly increased.
基金The National Key Research and Development Program of China(Grant No.2021YFA0805900)the 2020 Research Program of Sanya Yazhou Bay Science and Technology City(Grant No.202002011)+1 种基金the National Natural Science Foundation of China(Grant No.32002180)the Key Research and Development Program of Hainan Province,China(Grant No.ZDYF2021SHFZ230)。
文摘Animal models of human diseases play a critical role in medical research.Pigs are anatomically and physiologically more like humans than are small rodents such as mice,making pigs an attractive option for modeling human diseases.Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease.In the present review,we summarize the current status of pig models for human cardiovascular,metabolic,neurodegenerative,and various genetic diseases.We also discuss areas that need to be improved.Animal models of human diseases play a critical role in medical research.Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease.In the present review,we summarize the current status of pig models for human cardiovascular,metabolic,neurodegenerative,various genetic diseases and xenotransplantation.
文摘With supports from the National Natural Science Foundation of China and the National Key Research and Development Program,the research team led by Profs.Gao CaiXia(高彩霞)and Li JiaYang(李家洋)at the Institute of Genetics and Developmental Biology of Chinese Academy of Sciences and Associate Prof.Jiang LinJian(姜临建)at China Agricultural University,generated several herbicide-tolerant wheat germplasms using base editing to facilitate weed control in wheat fields.The paper,entitled“Generation of herbicide tolerance traits and a new selectable marker in wheat using base editing,”was published in Nature Plants(doi:10.1038/s41477-019-0405-0).
基金supported by the Key-Area Research and Development Program of Guangdong Province (2019B03035001)the National Natural Science Foundation of China (81941014, 31625013, 91732302, 81471312, 81771387, 81460352, 81500983, 31700897, 31700910, 31800901, 31700897, 31960178, and 81460352)+7 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences (XDBS32060200)the Shanghai Brain-Intelligence Project from the Science and Technology Commission of the Shanghai Municipality (16JC1420501)the Shanghai Municipal Science and Technology Major Project (2018SHZDZX05)the Applied Basic Research Programs of Science and Technology Commission Foundation of Yunnan Province (2017FB109, 2018FB052, 2018FB053, and 2019FA007)the China Postdoctoral Science Foundation (2018M631105)the CAS ‘‘Light of West China” Programthe National Key R&D Program of China (2018YFA0801403)the Key Scientific and Technological Projects of Guangdong Province (2018B030335001)。
基金This work was supported by the National Natural Science Foundation of China(81200368,81670160)the Hunan Natural Science Foundation(2017JJ2355).
文摘Background:T-cell acute lymphoblastic leukemia(T-ALL)is an uncommon and aggressive subtype of acute lymphoblastic leukemia(ALL).In the serum of T-ALL patients,the activity of lactate dehydrogenase A(LDHA)is increased.We proposed that targeting LDHA may be a potential strategy to improve T-ALL outcomes.The current study was conducted to investigate the antileukemic effect of LDHA gene-targeting treatment on T-ALL and the underlying molecular mechanism.Methods:Primary T-ALL cell lines Jurkat and DU528 were treated with the LDH inhibitor oxamate.MTT,colony formation,apoptosis,and cell cycle assays were performed to investigate the effects of oxamate on T-ALL cells.Quantitative real-time PCR(qPCR)and Western blotting analyses were applied to determine the related signaling pathways.A mitochondrial reactive oxygen species(ROS)assay was performed to evaluate ROS production after T-ALL cells were treated with oxamate.A T-ALL transgenic zebrafish model with LDHA gene knockdown was established using CRISPR/Cas9 gene-editing technology,and then TUNEL,Western blotting,and T-ALL tumor progression analyses were conducted to investigate the effects of LDHA gene knockdown on T-ALL transgenic zebrafish.Results:Oxamate significantly inhibited proliferation and induced apoptosis of Jurkat and DU528 cells.It also arrested Jurkat and DU528 cells in G0/G1 phase and stimulated ROS production(all P<0.001).Blocking LDHA significantly decreased the gene and protein expression of c-Myc,as well as the levels of phosphorylated serine/threonine kinase(AKT)and glycogen synthase kinase 3 beta(GSK-3β)in the phosphatidylinositol 3′-kinase(PI3K)signaling pathway.LDHA gene knockdown delayed disease progression and down-regulated c-Myc mRNA and protein expression in T-ALL transgenic zebrafish.Conclusion:Targeting LDHA exerted an antileukemic effect on T-ALL,representing a potential strategy for T-ALL treatment.
