As an essential crop that provides vegetable oil and protein,soybean(Glycine max(L.)Merr.)is widely planted all over the world.However,the scarcity of water resources worldwide has seriously impacted on the quality an...As an essential crop that provides vegetable oil and protein,soybean(Glycine max(L.)Merr.)is widely planted all over the world.However,the scarcity of water resources worldwide has seriously impacted on the quality and yield of soybean.To address this,exploring excellent genes for improving drought resistance in soybean is crucial.In this study,we identified natural variations of GmFNSII-2(flavone synthase II)significantly affect the drought resistance of soybeans.Through sequence analysis of GmFNSII-2 in 632 cultivated and 44 wild soybeans nine haplotypes were identified.The full-length allele GmFNSII-2^(C),but not the truncated allele GmFNSII-2^(A) possessing a nonsense nucleotide variation,increased enzyme activity.Further research found that GmDREB3,known to increase soybean drought resistance,bound to the promoter region of GmFNSII-2^(C).GmDREB3 positively regulated the expression of GmFNSII-2^(C),increased flavone synthase abundance and improved the drought resistance.Furthermore,a singlebase mutation in the GmFNSII-2^(C) promoter generated an additional drought response element(CCCCT),which had stronger interaction strength with GmDREB3 and increased its transcriptional activity under drought conditions.The frequency of drought-resistant soybean varieties with Hap 1(Pro:GmFNSII-2^(C))has increased,suggesting that this haplotype may be selected during soybean breeding.In summary,GmFNSII-2^(C) could be used for molecular breeding of drought-tolerant soybean.展开更多
Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For ...Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.展开更多
Base editors of the Cas9 system have been widely used for precise nucleotide substitution in crops. In this study, Cas12a was applied to construct plant cytosine base editors(CBEs). The main elements of Cas12aCBEs wer...Base editors of the Cas9 system have been widely used for precise nucleotide substitution in crops. In this study, Cas12a was applied to construct plant cytosine base editors(CBEs). The main elements of Cas12aCBEs were engineered and their efficiency was evaluated in stably transformed rice cells. An optimized ttCas12a-hyA3Bctd editor, consisting of a LbCas12a variant carrying catalytic inactive D832A and temperature-tolerance D156R double mutations, a truncated human APOBEC3B deaminase, a human RAD51 single-stranded DNA-binding domain, and double copies of UGI, outperformed other Cas12aCBEs in base editing efficiency. In T0transgenic rice plants, ttCas12a-hyA3Bctd edited an average of42.01% and a maximum of 68.75% of lines at six genomic targets. A-to-G conversions were generated in rice by an adenine base editor with a similar architecture to the optimized CBE. Our results provide preliminary evidence for the feasibility of robust and efficient plant Cas12a base editing systems, which could be useful for precise crop breeding.展开更多
The attacks on in-vehicle Controller Area Network(CAN)bus messages severely disrupt normal communication between vehicles.Therefore,researches on intrusion detection models for CAN have positive business value for veh...The attacks on in-vehicle Controller Area Network(CAN)bus messages severely disrupt normal communication between vehicles.Therefore,researches on intrusion detection models for CAN have positive business value for vehicle security,and the intrusion detection technology for CAN bus messages can effectively protect the invehicle network from unlawful attacks.Previous machine learning-based models are unable to effectively identify intrusive abnormal messages due to their inherent shortcomings.Hence,to address the shortcomings of the previous machine learning-based intrusion detection technique,we propose a novel method using Attention Mechanism and AutoEncoder for Intrusion Detection(AMAEID).The AMAEID model first converts the raw hexadecimal message data into binary format to obtain better input.Then the AMAEID model encodes and decodes the binary message data using a multi-layer denoising autoencoder model to obtain a hidden feature representation that can represent the potential features behind the message data at a deeper level.Finally,the AMAEID model uses the attention mechanism and the fully connected layer network to infer whether the message is an abnormal message or not.The experimental results with three evaluation metrics on a real in-vehicle CAN bus message dataset outperform some traditional machine learning algorithms,demonstrating the effectiveness of the AMAEID model.展开更多
The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) ...The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing.展开更多
The influence of rock mechanical properties on the electromagnetic radiation(EMR)mechanism of rock fracturing is an important research topic in solid mechanics and earthquake prediction.In this study,an EMR model of r...The influence of rock mechanical properties on the electromagnetic radiation(EMR)mechanism of rock fracturing is an important research topic in solid mechanics and earthquake prediction.In this study,an EMR model of rock fracturing considering the fracture factor,elastic modulus,Poisson’s ratio,radiation distance and crack length is derived based on the Hertz oscillator array assumption.An experimental system,including an electromagnetic shielding module,an EMR signal induction and transmission module,a signal recording module and a loading module,is developed to understand the EMR characteristics of four different rocks.The validity of the EMR theoretical model is verified and the relationships between the rock cracking morphology and the EMR waveform,amplitude and frequency are revealed.It is found that rock mechanical properties have obvious influences on the EMR waveform,amplitude and frequency during rock fracturing.This study provides a better understanding on the EMR mechanism of rock fracturing and can help to improve the accuracy of rock disaster prediction based on EMR.展开更多
Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths.However,targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCa...Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths.However,targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s.In this study,we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing.Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM.In addition,FrCas9 demonstrated activity against all 16 possible NNTA PAMs,achieving an efficiency of up to 35.3%in calli and generating homozygous or biallelic mutations in 31.3%of the T_(0)transgenic plants.A proof-ofconcept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content.Multiplex mutations and deletions were produced by bidirectional editing,mediated by FrCas9,using a single palindromic TATA sequence as a PAM.Moreover,we developed FrCas9-derived base editors capable of programmable conversion between AT and GC pairs in plants.This study highlights a versatile FrCas9 toolset for plant core promoter editing,offering great potential for the fine-tuning of gene expression and creating of new germplasms.展开更多
Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on...Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.展开更多
Dear Editor,Accurately labeling proteins in living plant cells has long been a challenge and can be addressed by targeted insertion of tag sequences in a given locus.Recent optimized plant prime editors(PEs)enable eff...Dear Editor,Accurately labeling proteins in living plant cells has long been a challenge and can be addressed by targeted insertion of tag sequences in a given locus.Recent optimized plant prime editors(PEs)enable efficient programmable installation of small insertions or deletions,including insertions of short sequences(Li et al.,2022a,2022b;Jiang et al.,2022;Xu et al.,2022;Zong et al.,2022;Zou et al.,2022).To investigate whether prime editing can be used to tag endogenous proteins in rice,we made use of the enpPE2 system described in our previous report(Li et al.,2022b).展开更多
Male sterility is an important trait in rice for hybrid rice(Oryza sativa)breeding.However,the factors involved in dominant male sterility are largely unknown.Here,we identified a gene from Sanming dominant genic male...Male sterility is an important trait in rice for hybrid rice(Oryza sativa)breeding.However,the factors involved in dominant male sterility are largely unknown.Here,we identified a gene from Sanming dominant genic male sterile rice,named Sanming dominant male sterility(SMS),and reported that an epi-allele of this locus contributes to male sterility.Segregation analysis attributed dominant male sterility to a single locus,SMS,which we characterized using a male-sterile near isogenic line(NIL)of rice cultivar 93-11.The SMS locus was heterozygous in the male-sterile 93-11 NIL,containing an epi-allele identical to that in 93-11,and an epi-allele identical to that in rice cultivar Nipponbare,which we refer to as SMS9 and SMSN,respectively.SMS9is silent and hyper-methylated,whereas SMSNis expressed and hypo-methylated in the 93-11 NIL.Overexpressing SMSNled to male sterility.Mutations in SMS rescued the male sterility of the 93-11 NIL.Interestingly,we observed the duplication of SMSN in Nipponbare,but did not observe the duplication of SMS9in 93-11.Together,these findings suggest that the reduced methylation and enhanced expression of the SMSNepi-allele in the 93-11 NIL is responsible for its role in conferring dominant male sterility.展开更多
Streptococcus pyogenes Cas9(SpCas9)is the most widely used genome editing tool in plants.The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif(PAM),significantly limiting its scope...Streptococcus pyogenes Cas9(SpCas9)is the most widely used genome editing tool in plants.The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif(PAM),significantly limiting its scope of application.Recently,five SpCas9 variants,SpCas9-NRRH,SpCas9-NRCH,SpCas9-NRTH,SpG,and SPRY,were developed to recognize non-canonical PAMs in human cells.In this study,these variants were engineered for plant genome editing,and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice(Oryza sativa)by stable transformation.Moreover,both cytosine base editors using a rat APOBEC1 or a human APO-BEC3a and adenine base editors using a directly evolved highly compatible TadA*-8e deaminase were developed from these SpCas9 variants.Our results demonstrated that the developed SpCas9 variantsbased base editors readily generated conversions between C.G and T.A in the target sites with noncanonical PAMs in transgenic rice lines.Collectively,the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.展开更多
An enhanced CDA-like(eCDAL)was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors(CT-CBE).Then,a novel plant dual-base editor vers...An enhanced CDA-like(eCDAL)was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors(CT-CBE).Then,a novel plant dual-base editor version1(pDuBE1)was developed by integrating TadA-8e into eCDAL.The editing efficiency of pDuBE1 could reach to 87.6%,with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7%in stably transformed plant cells.Our results showed that pDuBE1 could mediate robust dual editing in plant genome,providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.展开更多
Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a target...Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in rice.Our results showed that the pPE2 system could induce programmable editing at different genome sites.In transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures.To optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells.However,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing frequencies.Furthemore,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited cells.The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited cells.Taken together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.展开更多
Reactive oxygen species （ROS） are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the me...Reactive oxygen species （ROS） are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mecha- nisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY （SPY）, an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling~ SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signal- ing in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed.展开更多
Cytotoxic T cells targeting cancer neoantigens harboring driver mutations can lead to durable tumor regression in an HLAIdependent manner.However,it is difficult to extend the population of patients who are eligible f...Cytotoxic T cells targeting cancer neoantigens harboring driver mutations can lead to durable tumor regression in an HLAIdependent manner.However,it is difficult to extend the population of patients who are eligible for neoantigen-based immunotherapy,as immunogenic neoantigen-HLA pairs are rarely shared across different patients.Thus,a way to find other human leukocyte antigen(HLA)alleles that can also present a clinically effective neoantigen is needed.Recently,neoantigen-based immunotherapy targeting the KRAS G12D mutation in patients with HLA-C*08:02 has shown effectiveness.In a proof-ofconcept study,we proposed a combinatorial strategy(the combination of phylogenetic and structural analyses)to find potential HLA alleles that could also present KRAS G12D neoantigen.Compared to in silico binding prediction,this strategy avoids the uneven accuracy across different HLA alleles.Our findings extend the population of patients who are potentially eligible for immunotherapy targeting the KRAS G12D mutation.Additionally,we provide an alternative way to predict neoantigen-HLA pairs,which maximizes the clinical usage of shared neoantigens.展开更多
Organic light-emitting diodes(OLEDs)have drawn tremendous attention due to their widespread applications in flat-panel displays and solid-state lightings over the years[1-4].The charge injection is crucial for the per...Organic light-emitting diodes(OLEDs)have drawn tremendous attention due to their widespread applications in flat-panel displays and solid-state lightings over the years[1-4].The charge injection is crucial for the performance of OLEDs featuring sandwiched p-i-n structures[5-9].For OLEDs,an indium tin oxide(ITO)electrode with a work function of 4.7 eV,and most holetransporting materials(HTMs)with the highest occupied molecular orbitals(HOMOs)close to-5.5 eV.展开更多
基金supported by grants from the National Key Research and Development Program of China(2023YFF1000404,2022YFF10001501)the National Natural Science Foundation of China(32171971)。
文摘As an essential crop that provides vegetable oil and protein,soybean(Glycine max(L.)Merr.)is widely planted all over the world.However,the scarcity of water resources worldwide has seriously impacted on the quality and yield of soybean.To address this,exploring excellent genes for improving drought resistance in soybean is crucial.In this study,we identified natural variations of GmFNSII-2(flavone synthase II)significantly affect the drought resistance of soybeans.Through sequence analysis of GmFNSII-2 in 632 cultivated and 44 wild soybeans nine haplotypes were identified.The full-length allele GmFNSII-2^(C),but not the truncated allele GmFNSII-2^(A) possessing a nonsense nucleotide variation,increased enzyme activity.Further research found that GmDREB3,known to increase soybean drought resistance,bound to the promoter region of GmFNSII-2^(C).GmDREB3 positively regulated the expression of GmFNSII-2^(C),increased flavone synthase abundance and improved the drought resistance.Furthermore,a singlebase mutation in the GmFNSII-2^(C) promoter generated an additional drought response element(CCCCT),which had stronger interaction strength with GmDREB3 and increased its transcriptional activity under drought conditions.The frequency of drought-resistant soybean varieties with Hap 1(Pro:GmFNSII-2^(C))has increased,suggesting that this haplotype may be selected during soybean breeding.In summary,GmFNSII-2^(C) could be used for molecular breeding of drought-tolerant soybean.
基金the financial support of the China Agriculture Research System of MOF and MARA-Food Legumes(CARS-08)the Agricultural Science and Technology Innovation Program(ASTIP)of the Chinese Academy of Agricultural Sciences。
文摘Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.
基金funded by the National Natural Science Foundation of China (U19A2022 and 32000284)the Natural Science Foundation of Anhui Province (2208085Y11, 2108085Y07,2008085QC101, and 2008085MC71)+2 种基金the University Synergy Innovation Program of Anhui Province (GXXT-2021-056)Open Research Fund Program of Anhui Province Key Laboratory of Rice Genetics and Breeding (SDKF-2021-01 and SDKF-2022-04)Natural Science Research Project for Anhui Universities(KJ2021A0196)。
文摘Base editors of the Cas9 system have been widely used for precise nucleotide substitution in crops. In this study, Cas12a was applied to construct plant cytosine base editors(CBEs). The main elements of Cas12aCBEs were engineered and their efficiency was evaluated in stably transformed rice cells. An optimized ttCas12a-hyA3Bctd editor, consisting of a LbCas12a variant carrying catalytic inactive D832A and temperature-tolerance D156R double mutations, a truncated human APOBEC3B deaminase, a human RAD51 single-stranded DNA-binding domain, and double copies of UGI, outperformed other Cas12aCBEs in base editing efficiency. In T0transgenic rice plants, ttCas12a-hyA3Bctd edited an average of42.01% and a maximum of 68.75% of lines at six genomic targets. A-to-G conversions were generated in rice by an adenine base editor with a similar architecture to the optimized CBE. Our results provide preliminary evidence for the feasibility of robust and efficient plant Cas12a base editing systems, which could be useful for precise crop breeding.
基金supported by Chongqing Big Data Engineering Laboratory for Children,Chongqing Electronics Engineering Technology Research Center for Interactive Learning,Project of Science and Technology Research Program of Chongqing Education Commission of China. (No.KJZD-K201801601).
文摘The attacks on in-vehicle Controller Area Network(CAN)bus messages severely disrupt normal communication between vehicles.Therefore,researches on intrusion detection models for CAN have positive business value for vehicle security,and the intrusion detection technology for CAN bus messages can effectively protect the invehicle network from unlawful attacks.Previous machine learning-based models are unable to effectively identify intrusive abnormal messages due to their inherent shortcomings.Hence,to address the shortcomings of the previous machine learning-based intrusion detection technique,we propose a novel method using Attention Mechanism and AutoEncoder for Intrusion Detection(AMAEID).The AMAEID model first converts the raw hexadecimal message data into binary format to obtain better input.Then the AMAEID model encodes and decodes the binary message data using a multi-layer denoising autoencoder model to obtain a hidden feature representation that can represent the potential features behind the message data at a deeper level.Finally,the AMAEID model uses the attention mechanism and the fully connected layer network to infer whether the message is an abnormal message or not.The experimental results with three evaluation metrics on a real in-vehicle CAN bus message dataset outperform some traditional machine learning algorithms,demonstrating the effectiveness of the AMAEID model.
基金funded by the Genetically Modified Breeding Major Project(2016ZX08010-002-008)the National Natural Science Foundation of China(31701405)the Natural Science Foundation of Anhui Province,China(1708085QC60)。
文摘The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing.
基金supported by the National Natural Science Foundation of China(Grant Nos.51979146 and 11272178)。
文摘The influence of rock mechanical properties on the electromagnetic radiation(EMR)mechanism of rock fracturing is an important research topic in solid mechanics and earthquake prediction.In this study,an EMR model of rock fracturing considering the fracture factor,elastic modulus,Poisson’s ratio,radiation distance and crack length is derived based on the Hertz oscillator array assumption.An experimental system,including an electromagnetic shielding module,an EMR signal induction and transmission module,a signal recording module and a loading module,is developed to understand the EMR characteristics of four different rocks.The validity of the EMR theoretical model is verified and the relationships between the rock cracking morphology and the EMR waveform,amplitude and frequency are revealed.It is found that rock mechanical properties have obvious influences on the EMR waveform,amplitude and frequency during rock fracturing.This study provides a better understanding on the EMR mechanism of rock fracturing and can help to improve the accuracy of rock disaster prediction based on EMR.
基金funded by the Improved Varieties Joint Research(Rice)Project of Anhui Province(the 14th five-year plan),the National Key Research and Development Program(2022YFF1002803)the National Natural Science Foundation of China(No.32300343,32000284,and 32270430)+1 种基金the Natural Science Foundation of Anhui Province(No.2208085Y11 and 2308085Y20)the Innovative Research Team of Anhui Education(No.2022AH010056).
文摘Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths.However,targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s.In this study,we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing.Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM.In addition,FrCas9 demonstrated activity against all 16 possible NNTA PAMs,achieving an efficiency of up to 35.3%in calli and generating homozygous or biallelic mutations in 31.3%of the T_(0)transgenic plants.A proof-ofconcept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content.Multiplex mutations and deletions were produced by bidirectional editing,mediated by FrCas9,using a single palindromic TATA sequence as a PAM.Moreover,we developed FrCas9-derived base editors capable of programmable conversion between AT and GC pairs in plants.This study highlights a versatile FrCas9 toolset for plant core promoter editing,offering great potential for the fine-tuning of gene expression and creating of new germplasms.
基金supported by Genetically Modified Breeding Major Projects(No.2016ZX08010-002-008)the National Natural Science Foundation of China(Nos.31501239 and 31401454)
文摘Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.
基金funded by the National Key Research and Development Program(2022YFF1002803)the National Natural Science Foundation of China(U19A2022,32270430,and 32000284)+3 种基金the Natural Science Foundation of Anhui Province(2108085Y07,2208085Y11,and 2008085MC71)the Innovative Research Team of Anhui Education(2022AH010056)the Science and Technology Major Projects of Anhui Province(2021d06050002)the Improved Varieties Joint Research(Rice)Project of Anhui Province(the 14th 5-year plan).
文摘Dear Editor,Accurately labeling proteins in living plant cells has long been a challenge and can be addressed by targeted insertion of tag sequences in a given locus.Recent optimized plant prime editors(PEs)enable efficient programmable installation of small insertions or deletions,including insertions of short sequences(Li et al.,2022a,2022b;Jiang et al.,2022;Xu et al.,2022;Zong et al.,2022;Zou et al.,2022).To investigate whether prime editing can be used to tag endogenous proteins in rice,we made use of the enpPE2 system described in our previous report(Li et al.,2022b).
基金supported by the Global Select Project (DJK-LX-2022007)of the Institute of Health and Medicine,Hefei Comprehensive National Science Centerthe Key Research and Development Projects in Anhui Province (2023n06020035,202104b11020008)+3 种基金the University Synergy Innovation Program of Anhui Province (GXXT-2019033)the National Natural Science Foundation of China (U19A2021,32230017)the Anhui Province Major Special Project for Improving Seed Productionthe Strategic Priority Research Program“Molecular Mechanisms of Plant Growth and Development”of the Chinese Academy of Sciences (XDB27030203)。
文摘Male sterility is an important trait in rice for hybrid rice(Oryza sativa)breeding.However,the factors involved in dominant male sterility are largely unknown.Here,we identified a gene from Sanming dominant genic male sterile rice,named Sanming dominant male sterility(SMS),and reported that an epi-allele of this locus contributes to male sterility.Segregation analysis attributed dominant male sterility to a single locus,SMS,which we characterized using a male-sterile near isogenic line(NIL)of rice cultivar 93-11.The SMS locus was heterozygous in the male-sterile 93-11 NIL,containing an epi-allele identical to that in 93-11,and an epi-allele identical to that in rice cultivar Nipponbare,which we refer to as SMS9 and SMSN,respectively.SMS9is silent and hyper-methylated,whereas SMSNis expressed and hypo-methylated in the 93-11 NIL.Overexpressing SMSNled to male sterility.Mutations in SMS rescued the male sterility of the 93-11 NIL.Interestingly,we observed the duplication of SMSN in Nipponbare,but did not observe the duplication of SMS9in 93-11.Together,these findings suggest that the reduced methylation and enhanced expression of the SMSNepi-allele in the 93-11 NIL is responsible for its role in conferring dominant male sterility.
基金This work was funded by the Joint Project of China National Natural Science Foundation of China and Anhui province(no.U19A2022)Genetically Modified Breeding Major Projects(no.2016ZX08010-002-008 and no.2019ZX08010003-001-008)+3 种基金the National Natural Science Foundation(no.32000284)the Natural Science Foundation of Anhui Province(no.2008085QC101 and no.2008085MC71)the Key Technology Research Project of Hefei(J2020G44)Science and Technology Major Projects of Anhui Province(no.202003a06020009).
文摘Streptococcus pyogenes Cas9(SpCas9)is the most widely used genome editing tool in plants.The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif(PAM),significantly limiting its scope of application.Recently,five SpCas9 variants,SpCas9-NRRH,SpCas9-NRCH,SpCas9-NRTH,SpG,and SPRY,were developed to recognize non-canonical PAMs in human cells.In this study,these variants were engineered for plant genome editing,and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice(Oryza sativa)by stable transformation.Moreover,both cytosine base editors using a rat APOBEC1 or a human APO-BEC3a and adenine base editors using a directly evolved highly compatible TadA*-8e deaminase were developed from these SpCas9 variants.Our results demonstrated that the developed SpCas9 variantsbased base editors readily generated conversions between C.G and T.A in the target sites with noncanonical PAMs in transgenic rice lines.Collectively,the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.
基金funded by the National Natural Science Foundation of China(U19A2022 and 32000284)Natural Science Foundation of Anhui province(2008085QC101 and 2008085MC71)+2 种基金Open Research Fund Program of Anhui Province Key Laboratory of Rice Genetics and Breeding(SDKF-2021-04)Science and Technology Major Projects of Anhui Province(202003a06020009)Key Technology Research Project of Hefei(J2020G44)。
文摘An enhanced CDA-like(eCDAL)was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors(CT-CBE).Then,a novel plant dual-base editor version1(pDuBE1)was developed by integrating TadA-8e into eCDAL.The editing efficiency of pDuBE1 could reach to 87.6%,with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7%in stably transformed plant cells.Our results showed that pDuBE1 could mediate robust dual editing in plant genome,providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.
基金funded by the Genetically Modified Breeding Major Projects(no.2019ZX08010003-001-008 and no.2016ZX08010-002-008)the National Natural Science Foundation of China(no.U19A2022).
文摘Prime-editing systems have the capability to perform efficient and precise genome editing in human cells.In this study,we first developed a plant prime editor 2(pPE2)system and test its activity by generating a targeted mutation on an HPT^(-ATG) reporter in rice.Our results showed that the pPE2 system could induce programmable editing at different genome sites.In transgenic T0 plants,pPE2-generated mutants occurred with 0%–31.3%frequency,suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures.To optimize editing efficiency,guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells.However,at the genomic sites tested in this study,pPE3 systems generated only comparable or even lower editing frequencies.Furthemore,we developed a surrogate pPE2 system by incorporating the HPT^(-ATG) reporter to enrich the prime-edited cells.The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system,presumably due to the enhanced screening efficiency of edited cells.Taken together,our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.
文摘Reactive oxygen species （ROS） are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mecha- nisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY （SPY）, an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling~ SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signal- ing in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed.
基金supported by the National Natural Science Foundation of China(31870728,31470738,and 32000611)the National Basic Research Program of China(2014CB910103)+1 种基金the Fundamental Research Funds for the Central Universities(2042020kfxg02)the China Postdoctoral Science Foundation(2018M642918)。
文摘Cytotoxic T cells targeting cancer neoantigens harboring driver mutations can lead to durable tumor regression in an HLAIdependent manner.However,it is difficult to extend the population of patients who are eligible for neoantigen-based immunotherapy,as immunogenic neoantigen-HLA pairs are rarely shared across different patients.Thus,a way to find other human leukocyte antigen(HLA)alleles that can also present a clinically effective neoantigen is needed.Recently,neoantigen-based immunotherapy targeting the KRAS G12D mutation in patients with HLA-C*08:02 has shown effectiveness.In a proof-ofconcept study,we proposed a combinatorial strategy(the combination of phylogenetic and structural analyses)to find potential HLA alleles that could also present KRAS G12D neoantigen.Compared to in silico binding prediction,this strategy avoids the uneven accuracy across different HLA alleles.Our findings extend the population of patients who are potentially eligible for immunotherapy targeting the KRAS G12D mutation.Additionally,we provide an alternative way to predict neoantigen-HLA pairs,which maximizes the clinical usage of shared neoantigens.
基金supported by the National Key Basic Research and Development Program of China (2017YFA0204501 and 2020YFA0715000)the National Natural Science Foundation of China (51903137 and 61890942)+1 种基金Foshan Xianhu Laboratory of the Advanced Energy Science and Technology Guangdong Laboratory (XHT2020-005)financial support from the Young Elite Scientist Sponsorship Program (2019QNRC001) by China Association for Science and Technology。
文摘Organic light-emitting diodes(OLEDs)have drawn tremendous attention due to their widespread applications in flat-panel displays and solid-state lightings over the years[1-4].The charge injection is crucial for the performance of OLEDs featuring sandwiched p-i-n structures[5-9].For OLEDs,an indium tin oxide(ITO)electrode with a work function of 4.7 eV,and most holetransporting materials(HTMs)with the highest occupied molecular orbitals(HOMOs)close to-5.5 eV.
