Three-dimensional visualization technology converts engineering design drawings and data into graphics or images, realizes virtual reality perception of simulated users in future construction scene, enhances the inter...Three-dimensional visualization technology converts engineering design drawings and data into graphics or images, realizes virtual reality perception of simulated users in future construction scene, enhances the interaction between project management and technical personnel and engineering construction achievement, and provides intuitive, flexible and strong realistic experience for project management. It can effectively improve the level of project communication, and assist the needs of project construction planning management, training, exhibition, etc. As a tool to help improve project management skills, it has good application effect and prospects.展开更多
Dear Editor,This study reports a nuclease-mediated tiling deletion(NTD)method that uses LbCas12a nuclease with a tiling CRISPRderived RNA(crRNA)library to efficiently induce numerous nucleotide deletions in non-coding...Dear Editor,This study reports a nuclease-mediated tiling deletion(NTD)method that uses LbCas12a nuclease with a tiling CRISPRderived RNA(crRNA)library to efficiently induce numerous nucleotide deletions in non-coding regulatory regions of endogenous rice genes.This method was applied to non-coding regions of the Green Revolution gene SD1,generating 6 mutants with quantitative variations in plant height,which were then used to investigate associations between genotype and phenotype.NTD is thus a promising tool for molecular rice breeding.展开更多
CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors stri...CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.展开更多
Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for tr...Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.展开更多
Dual ion batteries(DIBs) exhibit broad application prospects in the field of electrical energy storage(EES)devices with excellent properties,such as high voltage,high energy density,and low cost.In the graphitebased D...Dual ion batteries(DIBs) exhibit broad application prospects in the field of electrical energy storage(EES)devices with excellent properties,such as high voltage,high energy density,and low cost.In the graphitebased DIBs,high voltage is needed to store enough anions with the formation of anion intercalation compound XCn(X=AlCl4-,PF6-,TFSI-,etc.).Hence,it is difficult for graphite-based DIBs to match proper anodes and electrolytes.Here,an Se/graphene composite is prepared via a convenient method,and assembled into a dual-ion full battery(DIFB) as anode with graphite cathode and 1 mol/L NaPF6 in EC:EMC(1:1,v:v).This DIFB has achieved a high discharge capacity of 75.9 mAh/g and high medium output voltage of 3.5 V at 0.1 A/g.Actually,the suitable anode materials,such as the present Se/graphene composite,are extremely important for the development and application of graphite-based DIBs.This study is enlightening for the design of future low-cost EES devices including graphite-based DIBs.展开更多
RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription.However,co-transcriptional splicing has not yet been explored in plants.Here,we built transcriptomes of nascent chromatin RNAs in A...RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription.However,co-transcriptional splicing has not yet been explored in plants.Here,we built transcriptomes of nascent chromatin RNAs in Arabidopsis thaliana and showed that nearly all introns undergo co-transcriptional splicing,which occurs with higher efficiency for introns in protein-coding genes than for those in noncoding RNAs.Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency,and introns with alternative 5′or 3′splice sites are less efficiently spliced.Furthermore,we found that mutations in genes encoding trans-acting proteins lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs,and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level.Collectively,our results not only uncovered widespread co-transcriptional splicing in Arabidopsis but also identified features that may affect or be affected by co-transcriptional splicing efficiency.展开更多
RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals.However,it is not known whether two other RNA quality control pathways,nonstop decay and no-go decay,are capable ...RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals.However,it is not known whether two other RNA quality control pathways,nonstop decay and no-go decay,are capable of restricting viruses in plants.Here,we show that the evolutionarily conserved Pelota–Hbs1 complex negatively regulates infection of plant viruses in the family Potyviridae(termed potyvirids),the largest group of plant RNA viruses that accounts for more than half of the viral crop damage worldwide.Pelota enables the recognition of the functional G1-2A6-7 motif in the P3 cistron,which is conserved in almost all potyvirids.This allows Pelota to target the virus and act as a viral restriction factor.Furthermore,Pelota interacts with the SUMO E2-conjugating enzyme SCE1 and is SUMOylated in planta.Blocking Pelota SUMOylation disrupts the ability to recruit Hbs1 and inhibits viral RNA degradation.These findings reveal the functional importance of Pelota SUMOylation during the infection of potyvirids in plants.展开更多
Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms con...Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.展开更多
CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas...CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate O s B Z R I gainof- function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.展开更多
Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding....Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.展开更多
文摘Three-dimensional visualization technology converts engineering design drawings and data into graphics or images, realizes virtual reality perception of simulated users in future construction scene, enhances the interaction between project management and technical personnel and engineering construction achievement, and provides intuitive, flexible and strong realistic experience for project management. It can effectively improve the level of project communication, and assist the needs of project construction planning management, training, exhibition, etc. As a tool to help improve project management skills, it has good application effect and prospects.
基金supported by grants from the National Natural Science Foundation of China,China (31871948)the STI 2030–Major Projects (2023ZD04074)+3 种基金the Hainan Yazhou Bay Seed Lab (B21HJ0215)the Central Public-interest Scientific Institution Basal Research Fund (Y2022QC03)the Key Research and Development Program of Shandong Province,China (Agricultural Seed Improvement Project,2022LZGC012)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences,China。
文摘Dear Editor,This study reports a nuclease-mediated tiling deletion(NTD)method that uses LbCas12a nuclease with a tiling CRISPRderived RNA(crRNA)library to efficiently induce numerous nucleotide deletions in non-coding regulatory regions of endogenous rice genes.This method was applied to non-coding regions of the Green Revolution gene SD1,generating 6 mutants with quantitative variations in plant height,which were then used to investigate associations between genotype and phenotype.NTD is thus a promising tool for molecular rice breeding.
基金supported by the STI 2030-Major Projects(2023ZD04074)the National Key Research and Development Program of China(2023YFD1202900)+2 种基金the Nanfan special project of the Chinese Academy of Agricultural Sciences(YBXM2313)the Hainan Seed Industry Laboratory(project of B23CJ0208)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.
基金supported by grants from the NIH GM114660 to Y.Zhaothe Research Grants Council of Hong Kong (CUHK466011,465112,466613,CUHK2/CRF/11G,C401114R and Ao E/M-05/12)+2 种基金NSFC/RGC (N_CUHK406/12)NSFC (31270226 and 31470294)Shenzhen Peacock Project (KQTD201101) to L.Jiang
文摘Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.
基金financial support by the National Natural Science Foundation of China(No.91963118)Fundamental Research Funds for the Central Universities(No.2412019ZD010)。
文摘Dual ion batteries(DIBs) exhibit broad application prospects in the field of electrical energy storage(EES)devices with excellent properties,such as high voltage,high energy density,and low cost.In the graphitebased DIBs,high voltage is needed to store enough anions with the formation of anion intercalation compound XCn(X=AlCl4-,PF6-,TFSI-,etc.).Hence,it is difficult for graphite-based DIBs to match proper anodes and electrolytes.Here,an Se/graphene composite is prepared via a convenient method,and assembled into a dual-ion full battery(DIFB) as anode with graphite cathode and 1 mol/L NaPF6 in EC:EMC(1:1,v:v).This DIFB has achieved a high discharge capacity of 75.9 mAh/g and high medium output voltage of 3.5 V at 0.1 A/g.Actually,the suitable anode materials,such as the present Se/graphene composite,are extremely important for the development and application of graphite-based DIBs.This study is enlightening for the design of future low-cost EES devices including graphite-based DIBs.
基金supported by the Fundamental Research Funds for the Central Universities,China and National Science Foundation of China(91740202).Y.W.was supported by a fellowship from Shenzhen Un iversity.
文摘RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription.However,co-transcriptional splicing has not yet been explored in plants.Here,we built transcriptomes of nascent chromatin RNAs in Arabidopsis thaliana and showed that nearly all introns undergo co-transcriptional splicing,which occurs with higher efficiency for introns in protein-coding genes than for those in noncoding RNAs.Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency,and introns with alternative 5′or 3′splice sites are less efficiently spliced.Furthermore,we found that mutations in genes encoding trans-acting proteins lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs,and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level.Collectively,our results not only uncovered widespread co-transcriptional splicing in Arabidopsis but also identified features that may affect or be affected by co-transcriptional splicing efficiency.
基金funded by the National Key Research and Development Program of China(2021YFD1400400)National Natural Science Foundation of China(31972244)to F.L.
文摘RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals.However,it is not known whether two other RNA quality control pathways,nonstop decay and no-go decay,are capable of restricting viruses in plants.Here,we show that the evolutionarily conserved Pelota–Hbs1 complex negatively regulates infection of plant viruses in the family Potyviridae(termed potyvirids),the largest group of plant RNA viruses that accounts for more than half of the viral crop damage worldwide.Pelota enables the recognition of the functional G1-2A6-7 motif in the P3 cistron,which is conserved in almost all potyvirids.This allows Pelota to target the virus and act as a viral restriction factor.Furthermore,Pelota interacts with the SUMO E2-conjugating enzyme SCE1 and is SUMOylated in planta.Blocking Pelota SUMOylation disrupts the ability to recruit Hbs1 and inhibits viral RNA degradation.These findings reveal the functional importance of Pelota SUMOylation during the infection of potyvirids in plants.
基金This work was supported by grants from the National Natural Science Foundation of China(31871948)the Fundamental Research Funds,and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences to H.Z.a grant from the Fundamental Research Funds for the Central Universities to S.L.
文摘Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.
基金National Natural Science Foundation of China (31871948)the National Key Research and Development Program of China (2017YFD0200900)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences to H.Z.
文摘CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate O s B Z R I gainof- function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.
基金supported by grants from the National Transgenic Science and Technology Program of China(2019ZX08010-003)to F.Y.the National Natural Science Foundation of China(31871948)the Fundamental Research Funds,and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(Y2020PT26)to H.Z.
文摘Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.
