Cotton is a pivotal economic crop for natural textile fibers that also serves as an important source of edible oil(Long et al.2023).Cottonseed oil contains approximately14%oleic acid and 59%linoleic acid.An increase i...Cotton is a pivotal economic crop for natural textile fibers that also serves as an important source of edible oil(Long et al.2023).Cottonseed oil contains approximately14%oleic acid and 59%linoleic acid.An increase in monounsaturated fatty acids,particularly oleic acid,enhances the oxidative stability and nutritional value of edible oil(Chen et al.2021).展开更多
The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of t...The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.展开更多
Objectives:Treatment of metastatic colorectal cancer(mCRC)includes resection of liver metastases(LM),however,no validated biomarker identifies patients most likely to benefit from this procedure.This meta-analysis aim...Objectives:Treatment of metastatic colorectal cancer(mCRC)includes resection of liver metastases(LM),however,no validated biomarker identifies patients most likely to benefit from this procedure.This meta-analysis aimed to assess the impact of the most relevant molecular alterations in cancer-related genes of CRC(i.e.,RAS,BRAF,SMAD4,PIK3CA)as prognostic markers of survival and disease recurrence in patients with mCRC surgically treated by LM resection.Methods:A systematic literature review was performed to identify studies reporting data regarding survival and/or recurrence in patients that underwent complete liver resection for CRC LM,stratified according to RAS,BRAF,PIK3CA,and SMAD4 mutational status.Hazard ratios(HRs)from multivariate analyses were pooled in the meta-analysis and various adjustment strategies for confounding factors were combined.The search was conducted in numerous databases,including MEDLINE(PubMed),Embase,Cumulative Index to Nursing and Allied Health Literature(CINAHL)(EBSCO host),and WHO Global Index Medicus,through March 18th,2022.Meta-analyses,editorials,letters to the editor,case reports,studies on other primary cancers,studies with primary metastatic sites other than the liver,studies lacking specific oncological outcome variables or genetic data,non-English language studies,and studies omitting residual disease data from liver metastasectomy were excluded.The remaining 47 studies were summarized in a descriptive table which outlines the key characteristics of each study and final results were graphically presented.Results:RAS mutation status was negatively associated with overall survival(OS)(HR,1.68;95%CI,1.54–1.84)and recurrence free survival(RFS)(HR,1.46;95%CI,1.33–1.61).A negative association was also found for BRAF regarding OS(HR,2.64;95%CI,2.15–3.24)and RFS(HR,1.89;95%CI,1.32–2.73)and SMAD4 regarding OS(HR,1.93;95%CI,1.56–2.38)and RFS(HR,1.95;95%CI,1.31–2.91).For PIK3CA only three studies were eligible and no significant association with either OS or RFS could be highlighted.Conclusion:RAS,BRAF,and SMAD4 are negatively associated with OS and RFS in patients undergoing curative liver metastasectomy from colorectal cancer.No conclusion can be drawn for PIK3CA due to the limited literature availability.These data support the integration of RAS,BRAF,and SMAD4 mutational status in the surgical decision-making for colorectal liver metastasis.Nevertheless,we have to consider several limitations,the major ones being the pooling of results from studies that evaluated patient outcomes as either disease-free survival(DFS)or RFS;the inclusion of patients with minimal residual disease and unconsidered potential confounding factors,such as variability in resectability definitions,chemotherapy use,and a potential interaction between biological markers and pre-and post-resection pharmacological treatments.展开更多
Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient trans...Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica,we systematically optimized factors affecting protoplast isolation and transient gene expression.We established an efficient protoplast-based transient gene expression system(PTGE)in Chinese cabbage,achieving high protoplast yield of 4.9×10^(5)·g^(-1)FW,viability over 95%,and transfection efficiency of 76%.We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency.Furthermore,protoplasts incubated at 37℃ for 6 min improved the transfection efficiency to 86%.We also demonstrated that PTGE worked well(more than 50%transfection efficiency)in multiple Brassica species including cabbage,Pak Choi,Chinese kale,and turnip.Finally,PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage,cabbage,and pak choi,demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.展开更多
Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most impo...Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.展开更多
The current major issue in improving detection sensitivity and selectivity is to design an electrochemical sensor that does not require PCR amplification for nucleic acid identification and measurement. Because of the...The current major issue in improving detection sensitivity and selectivity is to design an electrochemical sensor that does not require PCR amplification for nucleic acid identification and measurement. Because of their great sensitivity, precision, and simplicity of downsizing, electrochemical biosensors have emerged as a research hotspot in the field of nucleic acid detection. The CRISPR/Cas12 system has emerged as a potent tool for nucleic acid detection due to its powerful cleavage activity and selectivity. Specific electrode changes combined with the CRISPR/Cas12 system can greatly improve the performance of electrochemical biosensors. In this study, the design concepts of electrochemical biosensors based on the CRISPR/Cas12 system and their application advancements in nucleic acid detection are discussed.展开更多
Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in viv...Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in vivo models to study the role of Na^(+)/K^(+)-ATPase in these diseases,we modified the Drosophila gene homolog,Atpα,to mimic the human ATP1A1 gene mutations that cause CMT2.Mutations located within the helical linker region of human ATP1A1(I592T,A597T,P600T,and D601F)were simultaneously introduced into endogenous Drosophila Atpαby CRISPR/Cas9-mediated genome editing,generating the Atpα^(TTTF)model.In addition,the same strategy was used to generate the corresponding single point mutations in flies(Atpα^(I571T),Atpα^(A576T),Atpα^(P579T),and Atpα^(D580F)).Moreover,a deletion mutation(Atpα^(mut))that causes premature termination of translation was generated as a positive control.Of these alleles,we found two that could be maintained as homozygotes(Atpα^(I571T)and Atpα^(P579T)).Three alleles(Atpα^(A576T),Atpα^(P579)and Atpα^(D580F))can form heterozygotes with the Atpαmut allele.We found that the Atpαallele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila.Flies heterozygous for Atpα^(TTTF)mutations have motor performance defects,a reduced lifespan,seizures,and an abnormal neuronal morphology.These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.展开更多
基金supported by the Science and Technology Innovation Talents in Universities of Henan Province,China(24HASTIT053)the National Natural Science Foundation of China(32172041)+1 种基金the Natural Science Foundation of Henan Province,China(232300421026)the Science and Technology Innovation 2030,China(2022ZD0402001-04)。
文摘Cotton is a pivotal economic crop for natural textile fibers that also serves as an important source of edible oil(Long et al.2023).Cottonseed oil contains approximately14%oleic acid and 59%linoleic acid.An increase in monounsaturated fatty acids,particularly oleic acid,enhances the oxidative stability and nutritional value of edible oil(Chen et al.2021).
基金supported by the National Natural Science Foundation of China(32001532 and 31860411)the National Key Research and Development Program of China,(2022YFF1000020)+1 种基金Hunan Seed Industry Innovation Project(2021NK1012)the Yunnan Tobacco Company Project(2020530000241009)。
文摘The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.
基金partially funded by Italian Ministry of Health—Ricerca Corrente(no grant number).
文摘Objectives:Treatment of metastatic colorectal cancer(mCRC)includes resection of liver metastases(LM),however,no validated biomarker identifies patients most likely to benefit from this procedure.This meta-analysis aimed to assess the impact of the most relevant molecular alterations in cancer-related genes of CRC(i.e.,RAS,BRAF,SMAD4,PIK3CA)as prognostic markers of survival and disease recurrence in patients with mCRC surgically treated by LM resection.Methods:A systematic literature review was performed to identify studies reporting data regarding survival and/or recurrence in patients that underwent complete liver resection for CRC LM,stratified according to RAS,BRAF,PIK3CA,and SMAD4 mutational status.Hazard ratios(HRs)from multivariate analyses were pooled in the meta-analysis and various adjustment strategies for confounding factors were combined.The search was conducted in numerous databases,including MEDLINE(PubMed),Embase,Cumulative Index to Nursing and Allied Health Literature(CINAHL)(EBSCO host),and WHO Global Index Medicus,through March 18th,2022.Meta-analyses,editorials,letters to the editor,case reports,studies on other primary cancers,studies with primary metastatic sites other than the liver,studies lacking specific oncological outcome variables or genetic data,non-English language studies,and studies omitting residual disease data from liver metastasectomy were excluded.The remaining 47 studies were summarized in a descriptive table which outlines the key characteristics of each study and final results were graphically presented.Results:RAS mutation status was negatively associated with overall survival(OS)(HR,1.68;95%CI,1.54–1.84)and recurrence free survival(RFS)(HR,1.46;95%CI,1.33–1.61).A negative association was also found for BRAF regarding OS(HR,2.64;95%CI,2.15–3.24)and RFS(HR,1.89;95%CI,1.32–2.73)and SMAD4 regarding OS(HR,1.93;95%CI,1.56–2.38)and RFS(HR,1.95;95%CI,1.31–2.91).For PIK3CA only three studies were eligible and no significant association with either OS or RFS could be highlighted.Conclusion:RAS,BRAF,and SMAD4 are negatively associated with OS and RFS in patients undergoing curative liver metastasectomy from colorectal cancer.No conclusion can be drawn for PIK3CA due to the limited literature availability.These data support the integration of RAS,BRAF,and SMAD4 mutational status in the surgical decision-making for colorectal liver metastasis.Nevertheless,we have to consider several limitations,the major ones being the pooling of results from studies that evaluated patient outcomes as either disease-free survival(DFS)or RFS;the inclusion of patients with minimal residual disease and unconsidered potential confounding factors,such as variability in resectability definitions,chemotherapy use,and a potential interaction between biological markers and pre-and post-resection pharmacological treatments.
基金financially supported by the Key project of National Natural Science Foundation of China (Grant No.32330096)Innovative Research Group Project of Hebei Natural Science Foundation (Grant No.C2024204246)+3 种基金S&T Program of Hebei (Grant Nos.21372901D23567601H)Natural Science Foundation of Hebei (Grant No.C2023204119)the Starting Grant from Hebei Agricultural University (Grant No.YJ201958)。
文摘Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica,we systematically optimized factors affecting protoplast isolation and transient gene expression.We established an efficient protoplast-based transient gene expression system(PTGE)in Chinese cabbage,achieving high protoplast yield of 4.9×10^(5)·g^(-1)FW,viability over 95%,and transfection efficiency of 76%.We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency.Furthermore,protoplasts incubated at 37℃ for 6 min improved the transfection efficiency to 86%.We also demonstrated that PTGE worked well(more than 50%transfection efficiency)in multiple Brassica species including cabbage,Pak Choi,Chinese kale,and turnip.Finally,PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage,cabbage,and pak choi,demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.
基金supported by the Genomics Initiative of Agriculture and Agri-Food Canada。
文摘Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.
文摘The current major issue in improving detection sensitivity and selectivity is to design an electrochemical sensor that does not require PCR amplification for nucleic acid identification and measurement. Because of their great sensitivity, precision, and simplicity of downsizing, electrochemical biosensors have emerged as a research hotspot in the field of nucleic acid detection. The CRISPR/Cas12 system has emerged as a potent tool for nucleic acid detection due to its powerful cleavage activity and selectivity. Specific electrode changes combined with the CRISPR/Cas12 system can greatly improve the performance of electrochemical biosensors. In this study, the design concepts of electrochemical biosensors based on the CRISPR/Cas12 system and their application advancements in nucleic acid detection are discussed.
基金supported by the Natural Science Foundation of Fujian Province,No.2020J02027the National Natural Science Foundation of China,No.31970461the Foundation of NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-human Primate,Fujian Maternity and Child Health Hospital,No.2022-NHP-05(all to WC).
文摘Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in vivo models to study the role of Na^(+)/K^(+)-ATPase in these diseases,we modified the Drosophila gene homolog,Atpα,to mimic the human ATP1A1 gene mutations that cause CMT2.Mutations located within the helical linker region of human ATP1A1(I592T,A597T,P600T,and D601F)were simultaneously introduced into endogenous Drosophila Atpαby CRISPR/Cas9-mediated genome editing,generating the Atpα^(TTTF)model.In addition,the same strategy was used to generate the corresponding single point mutations in flies(Atpα^(I571T),Atpα^(A576T),Atpα^(P579T),and Atpα^(D580F)).Moreover,a deletion mutation(Atpα^(mut))that causes premature termination of translation was generated as a positive control.Of these alleles,we found two that could be maintained as homozygotes(Atpα^(I571T)and Atpα^(P579T)).Three alleles(Atpα^(A576T),Atpα^(P579)and Atpα^(D580F))can form heterozygotes with the Atpαmut allele.We found that the Atpαallele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila.Flies heterozygous for Atpα^(TTTF)mutations have motor performance defects,a reduced lifespan,seizures,and an abnormal neuronal morphology.These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.
