MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both acade...MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.展开更多
Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes.Although functional analyses of hundreds of these genes have been performed,there are still many t...Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes.Although functional analyses of hundreds of these genes have been performed,there are still many testis-enriched genes whose functions remain unexplored.Analyzing gene function using knockout(KO)mice is a powerful tool to discern if the gene of interest is essential for sperm formation,function,and male fertility in vivo.In this study,we generated KO mice for 12 testis-enriched genes,1700057G04Rik,4921539E11Rik,4930558C23Rik,Cby2,Ldhal6b,Rasef,Slc25a2,Slc25a41,Smim8,Smim9,Tmem210,and Tomm20l,using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation.Mating tests of KO mice reveal that these 12 genes are not essential for male fertility,at least when individually ablated,and not together with other potentially compensatory paralogous genes.Our results could prevent other laboratories from expending duplicative effort generating KO mice,for which no apparent phenotype exists.展开更多
Single-base editors,including cytosine base editors(CBEs)and adenine base editors(ABEs),facilitate accurate C·G to T·A and A·T to G·C,respectively,holding promise for the precise modeling and treat...Single-base editors,including cytosine base editors(CBEs)and adenine base editors(ABEs),facilitate accurate C·G to T·A and A·T to G·C,respectively,holding promise for the precise modeling and treatment of human hereditary disorders.Efficient base editing and expanded base conversion range have been achieved in human cells through base editors fusing with Rad51 DNA binding domain(Rad51DBD),such as hyA3A-BE4max.Here,we show that hyA3A-BE4max catalyzes C-to-T substitution in the zebrafish genome and extends editing positions(C_(12)-C_(16))proximal to the protospacer adjacent motif.We develop a codon-optimized counterpart zhyA3A-CBE5,which exhibits substantially high C-to-T conversion with 1.59-to 3.50-fold improvement compared with the original hyA3A-BE4max.With these tools,disease-relevant hereditary mutations can be more efficaciously generated in zebrafish.We introduce human genetic mutation rpl11^(Q42*)and abcc6a^(R1463C) by zhyA3A-CBE5 in zebrafish,mirroring Diamond-Blackfan anemia and Pseudoxanthoma Elasticum,respectively.Our study expands the base editing platform targeting the zebrafish genomic landscape and the application of single-base editors for disease modeling and gene function study.展开更多
Genome editing provides novel strategies for improving plant traits,but relies on current genetic transformation and plant regeneration procedures,which can be inefficient.We have engineered a barley stripe mosaic vir...Genome editing provides novel strategies for improving plant traits,but relies on current genetic transformation and plant regeneration procedures,which can be inefficient.We have engineered a barley stripe mosaic virus(BSMV)-based sgRNA delivery vector(BSMV-sg)that is effective in performing heritable genome editing in Cas9-transgenic wheat plants.Mutated progenies were present in the next generation at frequencies ranging from 12.9%to 100%in three different wheat varieties,and 53.8%to 100%of mutants were virus-free.We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs.Furthermore,we devised a virus-induced transgene-free editing procedure(VITF-Edit)to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat.Our study provides a robust,convenient and tissue culture-free approach for genome editing in wheat through virus infection.展开更多
Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of ...Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.展开更多
The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops.Here,we established efficient Agrobacterium-mediated clustered regularly...The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops.Here,we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)genome editing in a perennial,stress-tolerant forage grass,sheepgrass(Leymus chinensis).By screening for active single-guide RNAs(sg RNAs),accessions that regenerate well,suitable Agrobacterium strains,and optimal culture media,and co-expressing the morphogenic factor Ta WOX5,we achieved 11%transformation and 5.83%editing efficiency in sheepgrass.Knocking out Teosinte Branched1(TB1)significantly increased tiller number and biomass.This study opens avenues for studying gene function and breeding in sheepgrass.展开更多
Dear Editor,The CRISPR-Cas9（clustered regularly interspaced short palindromic repeats/Cas9）system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement a...Dear Editor,The CRISPR-Cas9（clustered regularly interspaced short palindromic repeats/Cas9）system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs（PAMs）.To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs and v variants with altered PAM specificities （Kleinstiver et al., 2015; Hu et al., 2016）.展开更多
Genome editing holds great promise for the molecular breeding of plants,yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants.Conventional plan...Genome editing holds great promise for the molecular breeding of plants,yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants.Conventional plant transformation-based methods for delivery of genome editing reagents into plants often involve prolonged tissue culture,a labor-intensive and technically challenging process for many elite crop cultivars.In this review,we describe various virus-based methods that have been employed to deliver genome editing reagents,including components of the CRISPR/Cas machinery and donor DNA for precision editing in plants.We update the progress in these methods with recent successful examples of genome editing achieved through virus-based delivery in different plant species,highlight the advantages and limitations of these delivery approaches,and discuss the remaining challenges.展开更多
CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable...CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.展开更多
It has been over a decade since the groundbreaking debut of CRISPR technology,and it's no exaggeration to say that it has revolutionized many areas of life sciences and biomedical research.CRISPR has significantly...It has been over a decade since the groundbreaking debut of CRISPR technology,and it's no exaggeration to say that it has revolutionized many areas of life sciences and biomedical research.CRISPR has significantly reduced the time required to generate animal models,opened new opportunities for developing new models on diverse species,accelerated the speed and expanded the scope of screening strategies,and most importantly,led to the development of new gene therapies for previously untreatable genetic diseases.展开更多
Following the publication of Liu et al.(2024),an error was identified in Figure 4B,in which the image representing the lung from the E529G group was inadvertently duplicated with the image of the lung from the WT grou...Following the publication of Liu et al.(2024),an error was identified in Figure 4B,in which the image representing the lung from the E529G group was inadvertently duplicated with the image of the lung from the WT group during figure preparation.展开更多
Signal transducer and activator of transcription 3(STAT3)is a member of the transcription factors involved in regulating many physiological and pathological processes,such as cell proliferation,angiogenesis and immune...Signal transducer and activator of transcription 3(STAT3)is a member of the transcription factors involved in regulating many physiological and pathological processes,such as cell proliferation,angiogenesis and immune escape.STAT3 has been identified as a potential therapeutic target for various cancers.Although numerous STAT3 inhibitors have been discovered and optimized to directly inhibit STAT3 activity,they are not yet authorized for clinical use and only a few have entered clinical trials.Furthermore,several proteolysis-targeting chimera(PROTAC)molecules with STAT3 degrading effects have been developed.The event-driven action of PROTAC overcome the drawbacks of STAT3,a traditional undruggable target,and addressed possible resistance to small-molecule inhibitors by degrading the entire STAT3 protein.In this review,we presented a brief introduction to STAT3 and its functions in various cancers,and systematically overviewed the pharmacological effects of inhibitors targeting different domains of STAT3 in the last three years,the structural characterization of the main scaffold,the design strategies,and the pharmacological activities of the substituents.Also,the binding patterns and interactions of some inhibitors with STAT3 were analyzed in detail and the recent advances in STAT3 degraders are also summarized.We anticipate that this perspective will contribute to the design and optimize more novel effective and specific STAT3 inhibitors or degraders for carcinoma treatment.展开更多
Transcription factors play critical roles in the regulation of gene expression during maize kernel development.The maize endosperm,a large storage organ,accounting for nearly 90%of the dry weight of mature kernels,ser...Transcription factors play critical roles in the regulation of gene expression during maize kernel development.The maize endosperm,a large storage organ,accounting for nearly 90%of the dry weight of mature kernels,serves as the primary site for starch storage.In this study,we identify an endosperm-specific EREB gene,ZmEREB167,which encodes a nucleus-localized EREB protein.Knockout of ZmEREB167 significantly increases kernel size and weight,as well as starch and protein content,compared with the wild type.In situ hybridization experiments show that ZmEREB167 is highly expressed in the BETL as well as PED regions of maize kernels.Dual-luciferase assays show that ZmEREB167 exhibits transcriptionally repressor activity in maize protoplasts.Transcriptome analysis reveals that a large number of genes are up-regulated in the Zmereb167-C1 mutant compared with the wild type,including key genetic factors such as ZmMRP-1 and ZmMN1,as well as multiple transporters involved in maize endosperm development.Integration of RNA-seq and ChIP-seq results identify 68 target genes modulated by ZmEREB167.We find that ZmEREB167 directly targets OPAQUE2,ZmNRT1.1,ZmIAA12,ZmIAA19,and ZmbZIP20,repressing their expressions.Our study demonstrates that ZmEREB167 functions as a negative regulator in maize endosperm development and affects starch accumulation and kernel size.展开更多
Tetracentron sinense is a‘living fossil’tree in East Asia.Understanding how this‘living fossil’responds to climate change and adapts to local environments is critical for its conservation.Here,we used resequenced ...Tetracentron sinense is a‘living fossil’tree in East Asia.Understanding how this‘living fossil’responds to climate change and adapts to local environments is critical for its conservation.Here,we used resequenced genomes to clarify the evolutionary history and adaptive potential of T.sinense.We identifiedsix divergent lineages in T.sinense:three lineages from southwestern China(Yunnan Province)and three lineages from the central subtropical region of China.Additionally,we detected hybridization events between some adjacent lineages.Demographic analysis revealed that over the past 10,000 years the effective population size(Ne)of three T.sinense lineages(i.e.,NORTH,SWEST,and YNWEST)increased after their last bottleneck and then remained stable,whereas that of the remaining three lineages(i.e.,YSEAST,YC,and EAST)declined steadily.The decline in effective population size in the Yunnan lineages aligned well with the decrease in genome-wide diversity and a significantincrease in the frequency of runs of homozygosity.Deleterious variants and positively selected sites were involved in the evolution of different lineages.Further,genotype–environment association(GEA)analyses indicated adaptation to temperature-and precipitation-related factors.Genomic offset analyses found the most vulnerable populations,while SC and SC-yad were predicted to better handle extreme changes.Our findingsprovide insights into the evolutionary history and conservation of T.sinense and enhance our understanding of the evolution of living fossil species.展开更多
Osteoarthritis(OA)is a common degenerative disease worldwide and new therapeutics that target inflammation and the crosstalk between immunocytes and chondrocytes are being developed to prevent and treat OA.These attem...Osteoarthritis(OA)is a common degenerative disease worldwide and new therapeutics that target inflammation and the crosstalk between immunocytes and chondrocytes are being developed to prevent and treat OA.These attempts involve repolarizing pro-inflammatory M1 macrophages into the anti-inflammatory M2 phenotype in synovium.In this study,we found that phosphoglycerate mutase 5(PGAM5)significantly increased in macrophages in OA synovium compared to controls based on histology of human samples and single-cell RNA sequencing results of mice models.To address the role of PGAM5 in macrophages in OA,we found conditional knockout of PGAM5 in macrophages greatly alleviated OA symptoms and promoted anabolic metabolism of chondrocytes in vitro and in vivo.Mechanistically,we found that PGAM5 enhanced M1 polarization via AKT-mTOR/p38/ERK pathways,whereas inhibited M2 polarization via STAT6-PPARγpathway in murine bone marrow-derived macrophages.Furthermore,we found that PGAM5 directly dephosphorylated Dishevelled Segment Polarity Protein 2(DVL2)which resulted in the inhibition ofβ-catenin and repolarization of M2 macrophages into M1 macrophages.Conditional knockout of both PGAM5 andβ-catenin in macrophages significantly exacerbated osteoarthritis compared to PGAM5-deficient mice.Motivated by these findings,we successfully designed mannose modified fluoropolymers combined with siPGAM5 to inhibit PGAM5 specifically in synovial macrophages via intra-articular injection,which possessed desired targeting abilities of synovial macrophages and greatly attenuated murine osteoarthritis.Collectively,these findings defined a key role for PGAM5 in orchestrating macrophage polarization and provides insights into novel macrophage-targeted strategy for treating OA.展开更多
CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing ce...CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing cells.To overcome the challenge,we report that a homology-independent targeted integration(HITI)strategy is used for permanent integration of high-specificity-activity Factor IX variant(F9 Padua,R338L)at the albumin(Alb)locus in a novel hemophilia B(HB)rat model.The knock-in efficiency reaches 3.66%,as determined by droplet digital PCR(dd PCR).The clotting time is reduced to a normal level four weeks after treatment,and the circulating factor IX(FIX)level is gradually increased up to 52%of the normal level over nine months even after partial hepatectomy,demonstrating the amelioration of hemophilia.Through primer-extension-mediated sequencing(PEM-seq),no significant off-target effect is detected.This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.展开更多
BACKGROUND Gut dysbiosis and changes in body composition(i.e.,a decrease in the proportion of muscle mass and an increase in extracellular fluid)are common in cirrhosis.AIM To study the relationship between the gut mi...BACKGROUND Gut dysbiosis and changes in body composition(i.e.,a decrease in the proportion of muscle mass and an increase in extracellular fluid)are common in cirrhosis.AIM To study the relationship between the gut microbiota and body composition in cirrhosis.METHODS This observational study included 46 patients with cirrhosis.Stool microbiome was assessed using 16S rRNA gene sequencing.Multifrequency bioelectrical impedance analysis was performed to assess body composition in these patients.RESULTS An increase in fat mass and a decrease in body cell mass were noted in 23/46(50.0%)and 15/46(32.6%)patients,respectively.Changes in the gut microbiome were not independently associated with the fat mass percentage in cirrhosis.The abundance of Bacteroidaceae(P=0.041)and Eggerthella(P=0.001)increased,whereas that of Erysipelatoclostridiaceae(P=0.006),Catenibacterium(P=0.021),Coprococcus(P=0.033),Desulfovibrio(P=0.043),Intestinimonas(P=0.028),and Senegalimassilia(P=0.015)decreased in the gut microbiome of patients with body cell mass deficiency.The amount of extracellular fluid increased in 22/46(47.6%)patients.Proteobacteria abundance(P<0.001)increased,whereas Firmicutes(P=0.023),Actinobacteria(P=0.026),Bacilli(P=0.008),Anaerovoraceceae(P=0.027),Christensenellaceae(P=0.038),Eggerthellaceae(P=0.047),Erysipelatoclostridiaceae(P=0.015),Erysipelotrichaceae(P=0.003),Oscillospiraceae(P=0.024),Rikenellaceae(P=0.002),Collinsella(P=0.030),Hungatella(P=0.040),Peptococcaceae(P=0.023),Slackia(P=0.008),and Senegalimassilia(P=0.024)abundance decreased in these patients.Patients with clinically significant ascites(n=9)had a higher abundance of Proteobacteria(P=0.031)and a lower abundance of Actinobacteria(P=0.019)and Bacteroidetes(P=0.046)than patients without clinically significant ascites(n=37).CONCLUSION Changes in the amount of body cell mass and extracellular fluid are associated with changes in the gut microbiome in cirrhosis patients.展开更多
Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants,such as tomato,papaya,cotton,and tobacco.Tomato yellow leaf curl virus(TYLCV)is a typical mo...Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants,such as tomato,papaya,cotton,and tobacco.Tomato yellow leaf curl virus(TYLCV)is a typical monopartite begomovirus that has been extensively studied,but methods that can efficiently control begomoviruses are still scarce.In this study,we combined artificial microRNA(amiRNA)-mediated silencing technology and clay nanosheetmediated delivery by spraying and developed a method for efficiently preventing TYLCV infection in tomato plants.We designed three amiRNAs that target different regions of TYLCV to silence virus-produced transcripts.Three plant expression vectors expressing pre-amiRNAs were constructed,and recombinant plasmid DNAs(pDNAs)were loaded onto nontoxic and degradable layered double hydroxide(LDH)clay nanosheets.LDH nanosheets containing multiple pDNAs were sprayed onto plant leaves.We found that the designed amiRNAs were significantly accumulated in leaves 7 days after spraying,while the pDNAs were sustainably detected for 35 days after the spray,suggesting that the LDH nanosheets released pDNAs in a sustained manner,protected pDNAs from degradation and efficiently delivered pDNAs into plant cells.Importantly,when the LDH nanosheets coated with pDNAs were sprayed onto plants infected by TYLCV,both the disease severity and TYLCV viral concentration in sprayed plants were significantly decreased during the 35 days,while the levels of H_(2)O_(2) were significantly increased in those plants.Taken together,these results indicate that LDH nanosheets loaded with pDNAs expressing amiRNAs can be a sustainable and promising tool for begomovirus control.展开更多
Trees in temperate regions exhibit evident seasonal patterns,which play vital roles in their growth and development.The activity of cambial stem cells is the basis for regulating the quantity and quality of wood,which...Trees in temperate regions exhibit evident seasonal patterns,which play vital roles in their growth and development.The activity of cambial stem cells is the basis for regulating the quantity and quality of wood,which has received considerable attention.However,the underlying mechanisms of these processes have not been fully elucidated.Here we performed a comprehensive analysis of morphological observations,transcriptome profiles,the DNA methylome,and miRNAs of the cambium in Populus tomentosa during the transition from dormancy to activation.Anatomical analysis showed that the active cambial zone exhibited a significant increase in the width and number of cell layers compared with those of the dormant and reactivating cambium.Furthermore,we found that differentially expressed genes associated with vascular development were mainly involved in plant hormone signal transduction,cell division and expansion,and cell wall biosynthesis.In addition,we identified 235 known miRNAs and 125 novel miRNAs.Differentially expressed miRNAs and target genes showed stronger negative correlations than other miRNA/target pairs.Moreover,global methylation and transcription analysis revealed that CG gene body methylation was positively correlated with gene expression,whereas CHG exhibited the opposite trend in the downstream region.Most importantly,we observed that the number of CHH differentially methylated region(DMR)changes was the greatest during cambium periodicity.Intriguingly,the genes with hypomethylated CHH DMRs in the promoter were involved in plant hormone signal transduction,phenylpropanoid biosynthesis,and plant–pathogen interactions during vascular cambium development.These findings improve our systems-level understanding of the epigenomic diversity that exists in the annual growth cycle of trees.展开更多
PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2...PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.展开更多
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020101 and XDA24020310)the National Natural Science Foundation of China(31672015,31788103)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003)。
文摘MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.
文摘Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes.Although functional analyses of hundreds of these genes have been performed,there are still many testis-enriched genes whose functions remain unexplored.Analyzing gene function using knockout(KO)mice is a powerful tool to discern if the gene of interest is essential for sperm formation,function,and male fertility in vivo.In this study,we generated KO mice for 12 testis-enriched genes,1700057G04Rik,4921539E11Rik,4930558C23Rik,Cby2,Ldhal6b,Rasef,Slc25a2,Slc25a41,Smim8,Smim9,Tmem210,and Tomm20l,using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation.Mating tests of KO mice reveal that these 12 genes are not essential for male fertility,at least when individually ablated,and not together with other potentially compensatory paralogous genes.Our results could prevent other laboratories from expending duplicative effort generating KO mice,for which no apparent phenotype exists.
基金supported by grants from Ministry of Science and Technology of the People's Republic of China(2018YFA0801004 and 2018YFA0800103)the National Natural Science Foundation of China(NSFC31530044,NSFC31970780,NSFC82202056).
文摘Single-base editors,including cytosine base editors(CBEs)and adenine base editors(ABEs),facilitate accurate C·G to T·A and A·T to G·C,respectively,holding promise for the precise modeling and treatment of human hereditary disorders.Efficient base editing and expanded base conversion range have been achieved in human cells through base editors fusing with Rad51 DNA binding domain(Rad51DBD),such as hyA3A-BE4max.Here,we show that hyA3A-BE4max catalyzes C-to-T substitution in the zebrafish genome and extends editing positions(C_(12)-C_(16))proximal to the protospacer adjacent motif.We develop a codon-optimized counterpart zhyA3A-CBE5,which exhibits substantially high C-to-T conversion with 1.59-to 3.50-fold improvement compared with the original hyA3A-BE4max.With these tools,disease-relevant hereditary mutations can be more efficaciously generated in zebrafish.We introduce human genetic mutation rpl11^(Q42*)and abcc6a^(R1463C) by zhyA3A-CBE5 in zebrafish,mirroring Diamond-Blackfan anemia and Pseudoxanthoma Elasticum,respectively.Our study expands the base editing platform targeting the zebrafish genomic landscape and the application of single-base editors for disease modeling and gene function study.
基金This work was supported by the Strategic Priority Research Program of the CAS(Precision Seed Design and Breeding,XDA24020310 and XDA24020100)the National Natural Science Foundation of China(31830106 and 31872637)+2 种基金The Project for Extramural Scientists of the State Key Laboratory of Agro-Biotechnology(2021SKLAB6-7)Chinese Universities Scientific Fund(2021TC112)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003).
文摘Genome editing provides novel strategies for improving plant traits,but relies on current genetic transformation and plant regeneration procedures,which can be inefficient.We have engineered a barley stripe mosaic virus(BSMV)-based sgRNA delivery vector(BSMV-sg)that is effective in performing heritable genome editing in Cas9-transgenic wheat plants.Mutated progenies were present in the next generation at frequencies ranging from 12.9%to 100%in three different wheat varieties,and 53.8%to 100%of mutants were virus-free.We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs.Furthermore,we devised a virus-induced transgene-free editing procedure(VITF-Edit)to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat.Our study provides a robust,convenient and tissue culture-free approach for genome editing in wheat through virus infection.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102 and XDA24010402)the National Natural Science Foundation of China (31788103 and 31971370)the Chinese Academy of Sciences (QYZDY-SSW-SMC030)
文摘Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.
基金supported by Key Projects in Science and Technology of Inner Mongolia (2021ZD0031)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA26030202)the Youth Innovation Promotion Association,CAS (2022096)。
文摘The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops.Here,we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)genome editing in a perennial,stress-tolerant forage grass,sheepgrass(Leymus chinensis).By screening for active single-guide RNAs(sg RNAs),accessions that regenerate well,suitable Agrobacterium strains,and optimal culture media,and co-expressing the morphogenic factor Ta WOX5,we achieved 11%transformation and 5.83%editing efficiency in sheepgrass.Knocking out Teosinte Branched1(TB1)significantly increased tiller number and biomass.This study opens avenues for studying gene function and breeding in sheepgrass.
基金supported by the National Key Research and Development Program of China (2016YFD0101800)the National Natural Science Foundation of China (91635301)+1 种基金the Zhejiang Provincial Natural Science Foundation of China (LZ14C130003)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences
文摘Dear Editor,The CRISPR-Cas9（clustered regularly interspaced short palindromic repeats/Cas9）system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs（PAMs）.To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs and v variants with altered PAM specificities （Kleinstiver et al., 2015; Hu et al., 2016）.
基金supported by STI 2030–Major Projects(2023ZD04074)the Natural Science Foundation of Jiangsu Province(BK20210384 and BK20212010)+4 种基金the Agricultural Science and Technology Innovation Program of Jiangsu Province[CX(22)3153]project of the Zhongshan Biological Breeding Laboratory(BM2022008-02)the Hainan Seed Industry Laboratory(B21HJ1004)the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University(NAUSY-ZZ01)the Jiangsu Specially Appointed Professor Program.
文摘Genome editing holds great promise for the molecular breeding of plants,yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants.Conventional plant transformation-based methods for delivery of genome editing reagents into plants often involve prolonged tissue culture,a labor-intensive and technically challenging process for many elite crop cultivars.In this review,we describe various virus-based methods that have been employed to deliver genome editing reagents,including components of the CRISPR/Cas machinery and donor DNA for precision editing in plants.We update the progress in these methods with recent successful examples of genome editing achieved through virus-based delivery in different plant species,highlight the advantages and limitations of these delivery approaches,and discuss the remaining challenges.
基金supported by the National Natural Science Foundation of China(31788103 and 31971370)the National Key Research and Development Program(2022YFF1002802)the Ministry of Agriculture and Rural Affairs of China,and the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020102).
文摘CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.
文摘It has been over a decade since the groundbreaking debut of CRISPR technology,and it's no exaggeration to say that it has revolutionized many areas of life sciences and biomedical research.CRISPR has significantly reduced the time required to generate animal models,opened new opportunities for developing new models on diverse species,accelerated the speed and expanded the scope of screening strategies,and most importantly,led to the development of new gene therapies for previously untreatable genetic diseases.
文摘Following the publication of Liu et al.(2024),an error was identified in Figure 4B,in which the image representing the lung from the E529G group was inadvertently duplicated with the image of the lung from the WT group during figure preparation.
基金supported by grants from the National Key Research and Development Program of China(No.2023YFA1800403)The Science and Technology Commission of Shanghai Municipality(No.21S11907800)Key Research and Development Program of Ningxia(No.2023BEG02010).
文摘Signal transducer and activator of transcription 3(STAT3)is a member of the transcription factors involved in regulating many physiological and pathological processes,such as cell proliferation,angiogenesis and immune escape.STAT3 has been identified as a potential therapeutic target for various cancers.Although numerous STAT3 inhibitors have been discovered and optimized to directly inhibit STAT3 activity,they are not yet authorized for clinical use and only a few have entered clinical trials.Furthermore,several proteolysis-targeting chimera(PROTAC)molecules with STAT3 degrading effects have been developed.The event-driven action of PROTAC overcome the drawbacks of STAT3,a traditional undruggable target,and addressed possible resistance to small-molecule inhibitors by degrading the entire STAT3 protein.In this review,we presented a brief introduction to STAT3 and its functions in various cancers,and systematically overviewed the pharmacological effects of inhibitors targeting different domains of STAT3 in the last three years,the structural characterization of the main scaffold,the design strategies,and the pharmacological activities of the substituents.Also,the binding patterns and interactions of some inhibitors with STAT3 were analyzed in detail and the recent advances in STAT3 degraders are also summarized.We anticipate that this perspective will contribute to the design and optimize more novel effective and specific STAT3 inhibitors or degraders for carcinoma treatment.
基金supported by STI 2030-Major Project(2023ZD04069)National Key Research and Development Program of China(2023YFD1202900)+3 种基金The National Science Fund for Distinguished Young Scholars(32425041)The“Breakthrough”Science and Technology Project of Tongliao(TL2024TW001)Science and Technology Demonstration Project of Shandong Province(2024SFGC0402)Pinduoduo-China Agricultural University Research Fund(PC2023A01004).
文摘Transcription factors play critical roles in the regulation of gene expression during maize kernel development.The maize endosperm,a large storage organ,accounting for nearly 90%of the dry weight of mature kernels,serves as the primary site for starch storage.In this study,we identify an endosperm-specific EREB gene,ZmEREB167,which encodes a nucleus-localized EREB protein.Knockout of ZmEREB167 significantly increases kernel size and weight,as well as starch and protein content,compared with the wild type.In situ hybridization experiments show that ZmEREB167 is highly expressed in the BETL as well as PED regions of maize kernels.Dual-luciferase assays show that ZmEREB167 exhibits transcriptionally repressor activity in maize protoplasts.Transcriptome analysis reveals that a large number of genes are up-regulated in the Zmereb167-C1 mutant compared with the wild type,including key genetic factors such as ZmMRP-1 and ZmMN1,as well as multiple transporters involved in maize endosperm development.Integration of RNA-seq and ChIP-seq results identify 68 target genes modulated by ZmEREB167.We find that ZmEREB167 directly targets OPAQUE2,ZmNRT1.1,ZmIAA12,ZmIAA19,and ZmbZIP20,repressing their expressions.Our study demonstrates that ZmEREB167 functions as a negative regulator in maize endosperm development and affects starch accumulation and kernel size.
基金supported by the National Natural Science Foundation of China(no.32570426)the Key Basic Research Program of Yunnan Province,China(202101BC070003)+1 种基金the Fundamental Research Funds for the Central Universities(QNTD202502)the STI 2030—Major Program(2022ZD0401605-2).
文摘Tetracentron sinense is a‘living fossil’tree in East Asia.Understanding how this‘living fossil’responds to climate change and adapts to local environments is critical for its conservation.Here,we used resequenced genomes to clarify the evolutionary history and adaptive potential of T.sinense.We identifiedsix divergent lineages in T.sinense:three lineages from southwestern China(Yunnan Province)and three lineages from the central subtropical region of China.Additionally,we detected hybridization events between some adjacent lineages.Demographic analysis revealed that over the past 10,000 years the effective population size(Ne)of three T.sinense lineages(i.e.,NORTH,SWEST,and YNWEST)increased after their last bottleneck and then remained stable,whereas that of the remaining three lineages(i.e.,YSEAST,YC,and EAST)declined steadily.The decline in effective population size in the Yunnan lineages aligned well with the decrease in genome-wide diversity and a significantincrease in the frequency of runs of homozygosity.Deleterious variants and positively selected sites were involved in the evolution of different lineages.Further,genotype–environment association(GEA)analyses indicated adaptation to temperature-and precipitation-related factors.Genomic offset analyses found the most vulnerable populations,while SC and SC-yad were predicted to better handle extreme changes.Our findingsprovide insights into the evolutionary history and conservation of T.sinense and enhance our understanding of the evolution of living fossil species.
基金This work was supported by grants from National Natural Science Foundation of China(81830078,82071868,32370892)Science and Technology Commission of Shanghai Municipality(23141901200)+2 种基金Health Commission of Shanghai Municipality(2022JC029)Biomaterials and Regenerative Medicine Institute Cooperative Research Project,Shanghai Jiaotong University School of Medicine(2022LHA11)Shanghai Key Laboratory of Orthopedic Implant(No.KFKT202206).
文摘Osteoarthritis(OA)is a common degenerative disease worldwide and new therapeutics that target inflammation and the crosstalk between immunocytes and chondrocytes are being developed to prevent and treat OA.These attempts involve repolarizing pro-inflammatory M1 macrophages into the anti-inflammatory M2 phenotype in synovium.In this study,we found that phosphoglycerate mutase 5(PGAM5)significantly increased in macrophages in OA synovium compared to controls based on histology of human samples and single-cell RNA sequencing results of mice models.To address the role of PGAM5 in macrophages in OA,we found conditional knockout of PGAM5 in macrophages greatly alleviated OA symptoms and promoted anabolic metabolism of chondrocytes in vitro and in vivo.Mechanistically,we found that PGAM5 enhanced M1 polarization via AKT-mTOR/p38/ERK pathways,whereas inhibited M2 polarization via STAT6-PPARγpathway in murine bone marrow-derived macrophages.Furthermore,we found that PGAM5 directly dephosphorylated Dishevelled Segment Polarity Protein 2(DVL2)which resulted in the inhibition ofβ-catenin and repolarization of M2 macrophages into M1 macrophages.Conditional knockout of both PGAM5 andβ-catenin in macrophages significantly exacerbated osteoarthritis compared to PGAM5-deficient mice.Motivated by these findings,we successfully designed mannose modified fluoropolymers combined with siPGAM5 to inhibit PGAM5 specifically in synovial macrophages via intra-articular injection,which possessed desired targeting abilities of synovial macrophages and greatly attenuated murine osteoarthritis.Collectively,these findings defined a key role for PGAM5 in orchestrating macrophage polarization and provides insights into novel macrophage-targeted strategy for treating OA.
基金supported by grants from National Key R&D Program of China(2019YFA0110802 and 2019YFA0802800)the National Natural Science Foundation of China(32025023,31971366)+1 种基金grants from the Shanghai Municipal Commission for Science and Technology(21CJ1402200,20140900200)the Innovation Program of Shanghai Municipal Education Commission(2019-01-07-00-05-E00054)。
文摘CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing cells.To overcome the challenge,we report that a homology-independent targeted integration(HITI)strategy is used for permanent integration of high-specificity-activity Factor IX variant(F9 Padua,R338L)at the albumin(Alb)locus in a novel hemophilia B(HB)rat model.The knock-in efficiency reaches 3.66%,as determined by droplet digital PCR(dd PCR).The clotting time is reduced to a normal level four weeks after treatment,and the circulating factor IX(FIX)level is gradually increased up to 52%of the normal level over nine months even after partial hepatectomy,demonstrating the amelioration of hemophilia.Through primer-extension-mediated sequencing(PEM-seq),no significant off-target effect is detected.This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.
基金Supported by Biocodex Microbiota Foundation:National Research Grant Russia 2019.
文摘BACKGROUND Gut dysbiosis and changes in body composition(i.e.,a decrease in the proportion of muscle mass and an increase in extracellular fluid)are common in cirrhosis.AIM To study the relationship between the gut microbiota and body composition in cirrhosis.METHODS This observational study included 46 patients with cirrhosis.Stool microbiome was assessed using 16S rRNA gene sequencing.Multifrequency bioelectrical impedance analysis was performed to assess body composition in these patients.RESULTS An increase in fat mass and a decrease in body cell mass were noted in 23/46(50.0%)and 15/46(32.6%)patients,respectively.Changes in the gut microbiome were not independently associated with the fat mass percentage in cirrhosis.The abundance of Bacteroidaceae(P=0.041)and Eggerthella(P=0.001)increased,whereas that of Erysipelatoclostridiaceae(P=0.006),Catenibacterium(P=0.021),Coprococcus(P=0.033),Desulfovibrio(P=0.043),Intestinimonas(P=0.028),and Senegalimassilia(P=0.015)decreased in the gut microbiome of patients with body cell mass deficiency.The amount of extracellular fluid increased in 22/46(47.6%)patients.Proteobacteria abundance(P<0.001)increased,whereas Firmicutes(P=0.023),Actinobacteria(P=0.026),Bacilli(P=0.008),Anaerovoraceceae(P=0.027),Christensenellaceae(P=0.038),Eggerthellaceae(P=0.047),Erysipelatoclostridiaceae(P=0.015),Erysipelotrichaceae(P=0.003),Oscillospiraceae(P=0.024),Rikenellaceae(P=0.002),Collinsella(P=0.030),Hungatella(P=0.040),Peptococcaceae(P=0.023),Slackia(P=0.008),and Senegalimassilia(P=0.024)abundance decreased in these patients.Patients with clinically significant ascites(n=9)had a higher abundance of Proteobacteria(P=0.031)and a lower abundance of Actinobacteria(P=0.019)and Bacteroidetes(P=0.046)than patients without clinically significant ascites(n=37).CONCLUSION Changes in the amount of body cell mass and extracellular fluid are associated with changes in the gut microbiome in cirrhosis patients.
基金funded by the National Natural Science Foundation of China(31801707)the Key Projects of Science and Technology Research in Henan Province(182102110470)+3 种基金the Plant Protection of Key Discipline Project of Henan province(107020219001/005)the National Key Research and Development Program of China(2016YFD0300203-3)First-Class Postdoctoral Research Grant in Henan Province(001701038)the Science-Technology Foundation for High Level Talent of Henan Institute of Science and Technology(2015028).
文摘Whitefly-transmitted begomoviruses are economically important plant pathogens that cause severe problems in many crop plants,such as tomato,papaya,cotton,and tobacco.Tomato yellow leaf curl virus(TYLCV)is a typical monopartite begomovirus that has been extensively studied,but methods that can efficiently control begomoviruses are still scarce.In this study,we combined artificial microRNA(amiRNA)-mediated silencing technology and clay nanosheetmediated delivery by spraying and developed a method for efficiently preventing TYLCV infection in tomato plants.We designed three amiRNAs that target different regions of TYLCV to silence virus-produced transcripts.Three plant expression vectors expressing pre-amiRNAs were constructed,and recombinant plasmid DNAs(pDNAs)were loaded onto nontoxic and degradable layered double hydroxide(LDH)clay nanosheets.LDH nanosheets containing multiple pDNAs were sprayed onto plant leaves.We found that the designed amiRNAs were significantly accumulated in leaves 7 days after spraying,while the pDNAs were sustainably detected for 35 days after the spray,suggesting that the LDH nanosheets released pDNAs in a sustained manner,protected pDNAs from degradation and efficiently delivered pDNAs into plant cells.Importantly,when the LDH nanosheets coated with pDNAs were sprayed onto plants infected by TYLCV,both the disease severity and TYLCV viral concentration in sprayed plants were significantly decreased during the 35 days,while the levels of H_(2)O_(2) were significantly increased in those plants.Taken together,these results indicate that LDH nanosheets loaded with pDNAs expressing amiRNAs can be a sustainable and promising tool for begomovirus control.
基金the State“13.5”Key Research Program of China(2016YFD0600102)the National Natural Science Foundation of China(31761133009,31970182,31670182,31800504,31970653,and 32000483)+2 种基金the Fundamental Research Funds for the Central Universities(2019ZY29 and 2017ZY10)Beijing Forestry University Outstanding Young Talent Cultivation Project(2019JQ03003)the Program of Introducing Talents of Discipline to Universities(111 project,B13007).
文摘Trees in temperate regions exhibit evident seasonal patterns,which play vital roles in their growth and development.The activity of cambial stem cells is the basis for regulating the quantity and quality of wood,which has received considerable attention.However,the underlying mechanisms of these processes have not been fully elucidated.Here we performed a comprehensive analysis of morphological observations,transcriptome profiles,the DNA methylome,and miRNAs of the cambium in Populus tomentosa during the transition from dormancy to activation.Anatomical analysis showed that the active cambial zone exhibited a significant increase in the width and number of cell layers compared with those of the dormant and reactivating cambium.Furthermore,we found that differentially expressed genes associated with vascular development were mainly involved in plant hormone signal transduction,cell division and expansion,and cell wall biosynthesis.In addition,we identified 235 known miRNAs and 125 novel miRNAs.Differentially expressed miRNAs and target genes showed stronger negative correlations than other miRNA/target pairs.Moreover,global methylation and transcription analysis revealed that CG gene body methylation was positively correlated with gene expression,whereas CHG exhibited the opposite trend in the downstream region.Most importantly,we observed that the number of CHH differentially methylated region(DMR)changes was the greatest during cambium periodicity.Intriguingly,the genes with hypomethylated CHH DMRs in the promoter were involved in plant hormone signal transduction,phenylpropanoid biosynthesis,and plant–pathogen interactions during vascular cambium development.These findings improve our systems-level understanding of the epigenomic diversity that exists in the annual growth cycle of trees.
基金National Key Research and Development Program of China,Grant/Award Number:2017YFD0500103National Natural Science Foundation of China,Grant/Award Number:31772747,31272385+3 种基金Jilin Province Science and Technology Development Projects,Grant/Award Number:20150204077NYGraduate Innovation Fund of Jilin Universitythe Program for Changjiang Scholarsthe University Innovative Research Team,Grant/Award Number:IRT1248
文摘PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.
