CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vec...CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high- efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/ Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edi- ted 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homol- ogous end-joining mechanism followed by homologous recombination-based repair. We also obtained uni- form biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mu- tations in To rice and T1Arabidopsis plants by simultaneous targeting of multiple （up to eight） members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.展开更多
Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing ...Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmlPKIA, ZmlPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmlPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies （13.1%） similar to those obtained with TALENs （9.1%）. Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.展开更多
Alzheimer’s disease （AD） is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved...Alzheimer’s disease （AD） is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved success in preclinical models addressing the pathological hallmarks of the disease, these efforts have not translated into any effective disease-modifying therapies. This could be because interventions are being tested too late in the disease process. While existing therapies provide symptomatic and clinical benefit, they do not fully address the molecular abnormalities that occur in AD neurons. The pathophysiology of AD is complex; mitochondrial bioenergetic deficits and brain hypometabolism coupled with increased mitochondrial oxidative stress are antecedent and potentially play a causal role in the disease pathogenesis. Dysfunctional mitochondria accumulate from the combination of impaired mitophagy, which can also induce injurious inflammatory responses, and inadequate neuronal mitochondrial biogenesis. Altering the metabolic capacity of the brain by modulating/potentiating its mitochondrial bioenergetics may be a strategy for disease prevention and treatment. We present insights into the mechanisms of mitochondrial dysfunction in AD brain as well as an overview of emerging treatments with the potential to prevent, delay or reverse the neurodegenerative process by targeting mitochondria.展开更多
Chronic hepatitis B infection is caused by hepatitis B virus(HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA(ccc DNA) is the key to establish a persistent infection within hepatoc...Chronic hepatitis B infection is caused by hepatitis B virus(HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA(ccc DNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing ccc DNA reservoir. Therefore, the study of the molecular mechanism of ccc DNA formation is becoming a major focus of HBV research. This review summarizes the current advances in ccc DNA molecular biology and the latest studies on the elimination or inactivation of ccc DNA, including three major areas:(1) epigenetic regulation of ccc DNA by HBV X protein,(2) immune-mediated degradation,and(3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.展开更多
Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treati...Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treating cancer. The problem is that RNAi, ASO, and ribozymes have poor stability and are constantly being degraded by nucleases. Researchers have made some efforts to increase antisense oligonucleotides’ stability by creating phospharimidate and Phosphorothioate. Currently, ribozymes, antisense oligonucleotides, and (RNAi) are the three main methods used to target RNA. These methods are currently undergoing clinical trials for the purpose of focusing on specific RNAs involved in disorders like cancer and neurodegeneration. In fact, ASOs that target amyotrophic lateral sclerosis and spinal muscular atrophy have produced promising results in clinical trials. The formation of chemical alterations that boost affinity and selectivity while reducing noxiousness owing to off-target impacts are two benefits of ASOs. Another benefit is increased affinity. With a focus on RNAi and ASOs, this review illustrated the main therapeutic strategies of RNA therapy now in use.展开更多
Mitochondrial diseases are a heterogeneous group of inherited disorders character-ized by mitochondrial dysfunction,and these diseases are often severe or even fatal.Mito-chondrial diseases are often caused by mitocho...Mitochondrial diseases are a heterogeneous group of inherited disorders character-ized by mitochondrial dysfunction,and these diseases are often severe or even fatal.Mito-chondrial diseases are often caused by mitochondrial DNA mutations.Currently,there is no curative treatment for patients with pathogenic mitochondrial DNA mutations.With the rapid development of traditional gene editing technologies,such as zinc finger nucleases and tran-scription activator-like effector nucleases methods,there has been a search for a mitochon-drial gene editing technology that can edit mutated mitochondrial DNA;however,there are still some problems hindering the application of these methods.The discovery of the DddA-derived cytosine base editor has provided hope for mitochondrial gene editing.In this paper,we will review the progress in the research on several mitochondrial gene editing technologies with the hope that this review will be useful for further research on mitochondrial gene editing technologies to optimize the treatment of mitochondrial diseases in the future.展开更多
Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit...Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.展开更多
Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors （TALEs） contain a DNA binding domain consist...Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors （TALEs） contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases （TALENs）, constructed using a method called ＂unit assembly＂, specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non- homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.展开更多
During the diversification of angiosperms, seeds have evolved structuralp chemical, molecular and physiolog- ically developing changes that specially affect the nucellus and endosperm. All through seed evolution, prog...During the diversification of angiosperms, seeds have evolved structuralp chemical, molecular and physiolog- ically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed celldeath （PCD） has played a fundamental role. However, examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus, suspensor and endosperm in those representative examples of seeds studied to date.展开更多
Despite relative effectiveness of current hepatitis B therapies,there is still no curative agents available.The new emerging approaches hold promise to achieve cure and loss of hepatitis B surface antigen.Studies or c...Despite relative effectiveness of current hepatitis B therapies,there is still no curative agents available.The new emerging approaches hold promise to achieve cure and loss of hepatitis B surface antigen.Studies or clinical trials investigating new therapies remain small and either focus on patients with low viral load and without hepatotoxic injury or patients with hepatitis D co-infection,which makes it challenging to assess their effectiveness and side effect profile in hepatitis B population.展开更多
Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleav...Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications byinducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases havebeen employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies,biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbitsusing transcription activator-like effector nucleases, and a perspective of the field.展开更多
The present work revealed that the praseodymium()complex of 2carboxyethylgermanium sesquioxide(Ge132)promotes the hydrolysis of the phosphodiester linkages of 3,5cyclic adenosine monophosphate(cAMP),3,5cyclic deoxyade...The present work revealed that the praseodymium()complex of 2carboxyethylgermanium sesquioxide(Ge132)promotes the hydrolysis of the phosphodiester linkages of 3,5cyclic adenosine monophosphate(cAMP),3,5cyclic deoxyadenosine monophosphate(dcAMP),5adenosine monophosphate(5AMP)and 5deoxyadenosine monophosphate(5dAMP)under mild conditions.Both cAMP and dcAMP were hydrolyzed sitespecifically,yielding predominantly 3monophosphates,the main products of the cleavage of 5AMP and 5dAMP included adenosine(Ado),deoxyadenosine(dAdo)and free phosphates respectively.A hydrolytic mechanism was proposed for cAMP,dcAMP,5AMP and 5dAMP.展开更多
Well-established targeted technologies to engi- neer genomes such as zinc-finger nuclease-based editing （ZFN）, transcription activator-like effector nuclease-based editing （TALEN）, and clustered regularly interspa...Well-established targeted technologies to engi- neer genomes such as zinc-finger nuclease-based editing （ZFN）, transcription activator-like effector nuclease-based editing （TALEN）, and clustered regularly interspaced short palindromic repeats and associated protein system-based editing （CRISPR/Cas） are proving to advance basic and applied research in numerous plant species. Compared with systems using ZFNs and TALENs, the most recently developed CRISPR/Cas system is more efficient due to its use of an RNA-guided nuclease to generate double-strand DNA breaks. To accelerate the applications of these technologies, we provide here a detailed overview of these systems, highlight the strengths and weaknesses of each, summarize research advances made with these technologies in model and crop plants, and discuss their applications in plant functional genomics. Such targeted approaches for genetically modifying plants will benefit agricultural production in the future.展开更多
Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading...Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.展开更多
Transcription activator-like effector （TALE） nucleases （TALENs） are increasingly used as a powerful tool for genome edit- ing in a variety of organisms. We have previously cloned the TALE-coding gene avrXa23 from ...Transcription activator-like effector （TALE） nucleases （TALENs） are increasingly used as a powerful tool for genome edit- ing in a variety of organisms. We have previously cloned the TALE-coding gene avrXa23 from Xanthomonas oryzae pv. oryzae and developed an AvrXa23-based assembly system for designer TALEs or TALENs. Here, we exploit TALENs to induce mutagenesis of the rice ethylene response factor （ERF） transcription factor OsERF922 for testing the gene-editing efficiency of AvrXa23-based TALENs system. A pair of TALENs （T-KJ9/KJ 10） was assembled and their nuclease activities were first confirmed in rice protoplast transient assay. The TALENs-expressing construct pT-KJ9/KJ10 was then used for rice transformation. We observed targeting somatic mutagenesis frequency of 15.0% in positive transgenic rice calli and obtained two mutant plants with nucleotide deletion or insertion at the designer target region. Our work demonstrates that the AvrXa23-based TALENs system can be used for site-specific genome editing in rice.展开更多
文摘CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high- efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/ Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edi- ted 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homol- ogous end-joining mechanism followed by homologous recombination-based repair. We also obtained uni- form biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mu- tations in To rice and T1Arabidopsis plants by simultaneous targeting of multiple （up to eight） members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.
基金supported by the National Natural Science Foundation of China (Grant Nos. 31271795 and 31200273)
文摘Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmlPKIA, ZmlPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmlPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies （13.1%） similar to those obtained with TALENs （9.1%）. Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.
文摘Alzheimer’s disease （AD） is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved success in preclinical models addressing the pathological hallmarks of the disease, these efforts have not translated into any effective disease-modifying therapies. This could be because interventions are being tested too late in the disease process. While existing therapies provide symptomatic and clinical benefit, they do not fully address the molecular abnormalities that occur in AD neurons. The pathophysiology of AD is complex; mitochondrial bioenergetic deficits and brain hypometabolism coupled with increased mitochondrial oxidative stress are antecedent and potentially play a causal role in the disease pathogenesis. Dysfunctional mitochondria accumulate from the combination of impaired mitophagy, which can also induce injurious inflammatory responses, and inadequate neuronal mitochondrial biogenesis. Altering the metabolic capacity of the brain by modulating/potentiating its mitochondrial bioenergetics may be a strategy for disease prevention and treatment. We present insights into the mechanisms of mitochondrial dysfunction in AD brain as well as an overview of emerging treatments with the potential to prevent, delay or reverse the neurodegenerative process by targeting mitochondria.
基金supported by the Key Project of Hubei Province Natural Science Foundation(2014CFA075)the National Natural Science Foundation of China(31400153)the Applied Basic Research Program(2015060101010033),Wuhan,China
文摘Chronic hepatitis B infection is caused by hepatitis B virus(HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA(ccc DNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing ccc DNA reservoir. Therefore, the study of the molecular mechanism of ccc DNA formation is becoming a major focus of HBV research. This review summarizes the current advances in ccc DNA molecular biology and the latest studies on the elimination or inactivation of ccc DNA, including three major areas:(1) epigenetic regulation of ccc DNA by HBV X protein,(2) immune-mediated degradation,and(3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.
文摘Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treating cancer. The problem is that RNAi, ASO, and ribozymes have poor stability and are constantly being degraded by nucleases. Researchers have made some efforts to increase antisense oligonucleotides’ stability by creating phospharimidate and Phosphorothioate. Currently, ribozymes, antisense oligonucleotides, and (RNAi) are the three main methods used to target RNA. These methods are currently undergoing clinical trials for the purpose of focusing on specific RNAs involved in disorders like cancer and neurodegeneration. In fact, ASOs that target amyotrophic lateral sclerosis and spinal muscular atrophy have produced promising results in clinical trials. The formation of chemical alterations that boost affinity and selectivity while reducing noxiousness owing to off-target impacts are two benefits of ASOs. Another benefit is increased affinity. With a focus on RNAi and ASOs, this review illustrated the main therapeutic strategies of RNA therapy now in use.
基金supported by the National Key R&D Program of China(No.2022YFA1104300,2021YFA1101902)the National Natural Science Foundation of China(No.82170364,82003756)+4 种基金the Natural Science Foundation of Jiangsu Province,China(No.BK20200800)China Postdoctoral Science Foundation(No.2022M712312)the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(No.21KJB310003)Jiangsu Funding Program for Excellent Postdoctoral Talent(China)(No.2022ZB577)Jiangsu Cardiovascular Medicine Innovation Center(China)(No.CXZX202210).
文摘Mitochondrial diseases are a heterogeneous group of inherited disorders character-ized by mitochondrial dysfunction,and these diseases are often severe or even fatal.Mito-chondrial diseases are often caused by mitochondrial DNA mutations.Currently,there is no curative treatment for patients with pathogenic mitochondrial DNA mutations.With the rapid development of traditional gene editing technologies,such as zinc finger nucleases and tran-scription activator-like effector nucleases methods,there has been a search for a mitochon-drial gene editing technology that can edit mutated mitochondrial DNA;however,there are still some problems hindering the application of these methods.The discovery of the DddA-derived cytosine base editor has provided hope for mitochondrial gene editing.In this paper,we will review the progress in the research on several mitochondrial gene editing technologies with the hope that this review will be useful for further research on mitochondrial gene editing technologies to optimize the treatment of mitochondrial diseases in the future.
文摘Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.
基金supported by grants from the National Basic Research Program of China (973 program, 2012CB113900)the National Natural Science Foundation of China (31071802)the Chongqing Natural Science Foundation (2011BA1002)
文摘Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors （TALEs） contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases （TALENs）, constructed using a method called ＂unit assembly＂, specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non- homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.
文摘During the diversification of angiosperms, seeds have evolved structuralp chemical, molecular and physiolog- ically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed celldeath （PCD） has played a fundamental role. However, examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus, suspensor and endosperm in those representative examples of seeds studied to date.
文摘Despite relative effectiveness of current hepatitis B therapies,there is still no curative agents available.The new emerging approaches hold promise to achieve cure and loss of hepatitis B surface antigen.Studies or clinical trials investigating new therapies remain small and either focus on patients with low viral load and without hepatotoxic injury or patients with hepatitis D co-infection,which makes it challenging to assess their effectiveness and side effect profile in hepatitis B population.
基金Work on this topic in the authors’laboratories is supported by grants from:the Strategic Priority Research Program of the Chinese Academy of Sciences(number XDA01020106)the Ministry of Science and Technology of China 973 program(2011CB965200)+2 种基金the National Natural Science Foundation of China(81261130317)to MAEthe Bureau of Science,Technology and Information of Guangzhou Municipality(2012 J5100040)to MAE and JFgrants 2010U1-E00811-5 and ZNGI-2011-010 from the Guangzhou Municipality and the Chinese Academy of Sciences,respectively,to LL.
文摘Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications byinducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases havebeen employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies,biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbitsusing transcription activator-like effector nucleases, and a perspective of the field.
文摘The present work revealed that the praseodymium()complex of 2carboxyethylgermanium sesquioxide(Ge132)promotes the hydrolysis of the phosphodiester linkages of 3,5cyclic adenosine monophosphate(cAMP),3,5cyclic deoxyadenosine monophosphate(dcAMP),5adenosine monophosphate(5AMP)and 5deoxyadenosine monophosphate(5dAMP)under mild conditions.Both cAMP and dcAMP were hydrolyzed sitespecifically,yielding predominantly 3monophosphates,the main products of the cleavage of 5AMP and 5dAMP included adenosine(Ado),deoxyadenosine(dAdo)and free phosphates respectively.A hydrolytic mechanism was proposed for cAMP,dcAMP,5AMP and 5dAMP.
文摘Well-established targeted technologies to engi- neer genomes such as zinc-finger nuclease-based editing （ZFN）, transcription activator-like effector nuclease-based editing （TALEN）, and clustered regularly interspaced short palindromic repeats and associated protein system-based editing （CRISPR/Cas） are proving to advance basic and applied research in numerous plant species. Compared with systems using ZFNs and TALENs, the most recently developed CRISPR/Cas system is more efficient due to its use of an RNA-guided nuclease to generate double-strand DNA breaks. To accelerate the applications of these technologies, we provide here a detailed overview of these systems, highlight the strengths and weaknesses of each, summarize research advances made with these technologies in model and crop plants, and discuss their applications in plant functional genomics. Such targeted approaches for genetically modifying plants will benefit agricultural production in the future.
文摘Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.
基金supported by the grant from the Major Science and Technology Project to Create New Crop Cultivars Using Gene Transfer Technology of China (2014ZX0801001B)the National Natural Science Foundation of China (31171812)
文摘Transcription activator-like effector （TALE） nucleases （TALENs） are increasingly used as a powerful tool for genome edit- ing in a variety of organisms. We have previously cloned the TALE-coding gene avrXa23 from Xanthomonas oryzae pv. oryzae and developed an AvrXa23-based assembly system for designer TALEs or TALENs. Here, we exploit TALENs to induce mutagenesis of the rice ethylene response factor （ERF） transcription factor OsERF922 for testing the gene-editing efficiency of AvrXa23-based TALENs system. A pair of TALENs （T-KJ9/KJ 10） was assembled and their nuclease activities were first confirmed in rice protoplast transient assay. The TALENs-expressing construct pT-KJ9/KJ10 was then used for rice transformation. We observed targeting somatic mutagenesis frequency of 15.0% in positive transgenic rice calli and obtained two mutant plants with nucleotide deletion or insertion at the designer target region. Our work demonstrates that the AvrXa23-based TALENs system can be used for site-specific genome editing in rice.
