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
Viruses evolve rapidly and continuously threaten animal health and economy,posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine.We presen...Viruses evolve rapidly and continuously threaten animal health and economy,posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine.We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase.We cloned pseudorabies virus genome into bacterial artificial chromosome,and used CRISPR-guided cytidine deaminase to directly convert cytidine(C)to uridine(U)to induce premature stop mutagenesis in viral genes.The editing efficiencies were 100%.Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus(PRV)genomes.Notably,in our study viral genome exists as a plasmid in E.coli,suggesting that this method is virus species-independent.This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.展开更多
New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable,and their development can be drastically accelerated by facile genetic manipulation methods in Mycob...New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable,and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis(M.tuberculosis).Clustered regularly interspaced short palindromic repeat(CRISPR)base editors allow for rapid,robust,and programmed single-base substitutions and gene inactivation,yet no such systems are currently available in M.tuberculosis.By screening distinct CRISPR base editors,we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9(St1Cas9)cytosine base editor(CBE)-but not the widely used Streptococcus pyogenes Cas9(SpCas9)or Lachnospiraceae bacterium Cpf1(LbCpf1)CBEs-is active in mycobacteria.Despite the notable C-to-T conversions,a high proportion of undesired byproducts exists with St1Cas9 CBE.We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor(UGI)or uracil DNA glycosylase(UNG)fusion,yielding two new base editors(CTBE and CGBE)capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis(M.smegmatis).Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif(PAM)sequence for DNA targeting,we engineered a PAM-expanded St1Cas9 variant by means of structureguided protein engineering for the base editors,substantially broadening the targeting scope.We first developed and characterized CTBE and CGBE in M.smegmatis,and then applied CTBE for genome editing in M.tuberculosis.Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics,antibiotic-resistant mechanism study,and drugtarget exploration in M.tuberculosis and related organisms.展开更多
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
The first editorial board meeting of the World Journal of Hepatology(WJH)was held on November 8,2019 at the Side Bar Grille,Sheraton Boston Hotel,Boston,MA,United States.Ruo-Yu Ma,Director of Editorial Office,on behal...The first editorial board meeting of the World Journal of Hepatology(WJH)was held on November 8,2019 at the Side Bar Grille,Sheraton Boston Hotel,Boston,MA,United States.Ruo-Yu Ma,Director of Editorial Office,on behalf of the Baishideng Publishing Group(BPG),organized the meeting with the great help of Professor Ke-Qin Hu,Journal Editor-in-Chief.There were six editorial board members,including two Editors-in-Chief and one administrative director of the editorial office at the meeting,discussing future strategies of the journal’s development.The editorial board provided BPG a number of suggestions in regard to the business plan and quality control of the WJH.Regarding the business aspect,the editorial board suggested that BPG should advertise the WJH at the international Hepatology and Gastroenterology conferences and promote the WJH via social media.On the scientific aspect,the editorial board suggested that the assessment systems for managing the reviewers and the editorial board members are necessary,and that the BPG should make efforts to attract more high-quality manuscript submissions.An additional comment was to continue to foster a scientific culture for the journal.In conclusion,it was noted that these new ideas expressed during the meeting will bring the WJH to the next level.In the future,the BPG and the editorial board will increase communication and collaboration in order to further the development of the WJH.展开更多
Statistical analysis of various data for the papers published in Journal of Shanghai University （ English Edition） from 1997 to 2004 was made. The issues examined include numbers of published papers, disciplinary di...Statistical analysis of various data for the papers published in Journal of Shanghai University （ English Edition） from 1997 to 2004 was made. The issues examined include numbers of published papers, disciplinary distribution, constituents of authors, propor- tions of collaborated papers contributed by multiple authors and papers based on cooperation between different institutions, publishing retardation, and citation of references. Suggestions are made for further improving the quality of the Journal.展开更多
Acta Geologica Sinica (formerly the Bulletin of the Geological Society of China) beganpublication in 1922, and was published mainly in English through 1952. Since then, thejournal has been published in Chinese with En...Acta Geologica Sinica (formerly the Bulletin of the Geological Society of China) beganpublication in 1922, and was published mainly in English through 1952. Since then, thejournal has been published in Chinese with English abstracts. In March 1988, ActaGeologica Sinica appeared in English language translation. The journal is a full translationof the Chinese edition, and follows the Chinese edition by two issues.展开更多
Acta Geologica Sinica (formerly the Bulletin of the Geological Society of China) beganpublication in 1922, and was published mainly in English through 1952. Since then, thejournal has been published in Chinese with En...Acta Geologica Sinica (formerly the Bulletin of the Geological Society of China) beganpublication in 1922, and was published mainly in English through 1952. Since then, thejournal has been published in Chinese with English abstracts. In March 1988, Acta展开更多
Dr.Chunmiao Zheng,a deputy editor-in-chief for Acta Geologica Sinica（English Edition）,received the 2013 O.E.Meinzer Award from the Geological Society of America.Dr.Zheng was also awarded the 2013 M.King Hubbert Awar...Dr.Chunmiao Zheng,a deputy editor-in-chief for Acta Geologica Sinica（English Edition）,received the 2013 O.E.Meinzer Award from the Geological Society of America.Dr.Zheng was also awarded the 2013 M.King Hubbert Award by the National Ground Water Association in the United States.＂It is quite an achievement to receive both of these awards in a lifetime - let alone in the same year!＂ said Professor Mary Anderson of the University of Wisconsin-Madison, a member of the US National Academy of Engineering and Dr. Zheng＇s Ph.D. advisor. Dr. Zheng is currently Chair Professor and Director of the Institute of Water Sciences at Peking University. He has also been the George Lindahl llI Endowed Professor of Hydrogeology at the University of Alabama, United States.展开更多
Soybean is the primary source of plant protein for humans.Owing to the indigestibility of the raffinose family of oligosaccharides(RFO),raffinose and stachyose are considered anti-nutritive factors in soybean seeds.Lo...Soybean is the primary source of plant protein for humans.Owing to the indigestibility of the raffinose family of oligosaccharides(RFO),raffinose and stachyose are considered anti-nutritive factors in soybean seeds.Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes,but the carbohydrate profiles invite further modification to lower RFOs.This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis,encoding three raffinose synthases(RS2,RS3,and RS4)and one stachyose synthase.In T1progeny,rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant(rfo-4m)were characterized.The rs2/rs3 mutant showed reduced raffinose and stachyose contents,but the rs4/sts mutant showed only reduced stachyose in seeds.The RFO contents in the rfo-4m mutant were almost eliminated.Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds,including the accumulation of several oligosaccharides-related metabolites.These mutants could contribute to precision breeding of soybean cultivars for soy food production.展开更多
The innovation of CRISPR/Cas gene editing technology has developed rapidly in recent years.It is widely used in the fields of disease animal model construction,biological breeding,disease diagnosis and screening,gene ...The innovation of CRISPR/Cas gene editing technology has developed rapidly in recent years.It is widely used in the fields of disease animal model construction,biological breeding,disease diagnosis and screening,gene therapy,cell localization,cell lineage tracking,synthetic biology,information storage,etc.However,developing idealized editors in various fields is still a goal for future development.This article focuses on the development and innovation of non-DSB editors BE and PE in the platform-based CRISPR system.It first explains the application of ideas for improvement such as“substitution”,“combination”,“adaptation”,and“adjustment”in BE and PE development and then catalogues the ingenious inversions and leaps of thought reflected in the innovations made to CRISPR technology.It will then elaborate on the efforts currently being made to develop small editors to solve the problem of AAV overload and summarize the current application status of editors for in vivo gene modification using AAV as a delivery system.Finally,it summarizes the inspiration brought by CRISPR/Cas innovation and assesses future prospects for development of an idealized editor.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
The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement.CRISPR/Cas12i3,which belongs to the ClassⅡTypeⅤ-...The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement.CRISPR/Cas12i3,which belongs to the ClassⅡTypeⅤ-ⅡCas system,has attracted extensive attention recently due to its smaller protein size and less restricted canonical"TTN"protospacer adjacent motif(PAM).However,due to its relatively lower editing efficiency,Cas12i3-mediated multiplex gene editing has not yet been documented in plants.Here,we fused four 5'exonucleases(Exo)including T5E,UL12,PapE,ME15 to the N terminal of an optimized Cas12i3 variant(Cas12i3-5M),respectively,and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines.We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M,respectively.Notably,the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61%across the six tested endogenous genes.We further demonstrated that,although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing,UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three,four,five or six genes with efficiencies of 82.76%,61.36%,52.94%,and 51.06%in rice stable lines,respectively.Together,we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice.The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.展开更多
CGBE(C-to-G base editor)systems,pivotal components within the base editing arsenal,enable the precise conversion of cytosines to guanines.However,conventional cytidine deaminases possess non-specific single-stranded D...CGBE(C-to-G base editor)systems,pivotal components within the base editing arsenal,enable the precise conversion of cytosines to guanines.However,conventional cytidine deaminases possess non-specific single-stranded DNA binding properties,leading to off-target effects and safety concerns.The Cas-embedding strategy,which involves embedding functional proteins like deaminases within the Cas9 enzyme’s architecture,emerges as a method to mitigate these off-target effects.Our study pioneers the application of the Cas-embedding strategy to CGBE systems,engineering a suite of novel CGBE editors,CE-CGBE.The CE-CGBE that incorporated eA3A,RBMX and Udgx excelled in editing efficiency,editing purity,and indel formation was named HF-CGBE.HF-CGBE showed no significant difference in off-target effects compared to the negative control group for both DNA and RNA.In summary,the novel HF-CGBE editors we propose expand the base editing toolbox and provide therapeutic approaches for related pathogenic mutations.展开更多
CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors stri...CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.展开更多
The programmable CRISPR-Cas genome editing technology,adopted from prokaryotic adaptive immune systems,has revolutionized genome engineering in plants(Liu et al.,2022a).Many efforts have been made to improve the activ...The programmable CRISPR-Cas genome editing technology,adopted from prokaryotic adaptive immune systems,has revolutionized genome engineering in plants(Liu et al.,2022a).Many efforts have been made to improve the activity,specificity,and protospacer adjacent motif(PAM)variants of Class 2 Cas nucleases,such as Cas9,Cas12a,and Cas12b(Liu et al.,2022a).However,their large size(∼1000–1400 amino acids)poses a challenge in scenarios requiring a compact Cas nuclease,particularly in urgent situations like plant virus-induced genome editing(Cheuk and Houde,2018;Li et al.,2021;Varanda et al.,2021).展开更多
Ongoing advances in genomics and genetics have led researchers to highlight the significant role of large DNA structural variants(SVs)in genetic diversity.SVs are genomic sequence variants of various types,including i...Ongoing advances in genomics and genetics have led researchers to highlight the significant role of large DNA structural variants(SVs)in genetic diversity.SVs are genomic sequence variants of various types,including insertions,deletions,inversions,translocations,and copy number variants(CNVs or duplications).These SVs,which are usually of 1 kb or more,can have profound impacts at both the individual and species level.They are also often linked to the initiation and progression of complex genetic diseases(Stankiewicz and Lupski,2010).展开更多
Primary hyperoxaluria type 1(PH1)is a severe hereditary disease,leading to the accumulation of oxalate in multiple organs,particularly the kidney.Hydroxyacid oxidase 1(HAO1),a pivotal gene involved in oxalate producti...Primary hyperoxaluria type 1(PH1)is a severe hereditary disease,leading to the accumulation of oxalate in multiple organs,particularly the kidney.Hydroxyacid oxidase 1(HAO1),a pivotal gene involved in oxalate production,is an approved target for the treatment of PH1.In this study,we demonstrated the discovery of several novel therapeutic sites of the Hao1 gene and the efficient editing of Hao1c.290-2 A in vivo with lipid nanoparticles(LNP)delivered adenine base editing(ABE)mRNA.A single infusion of LNP-ABE resulted in a near-complete knockout of Hao1 in the liver,leading to the sustainable normalization of urinary oxalate(for at least 6 months)and complete rescue of the pathophysiology in PH1 rats.Additionally,a significant correlation between Hao1 editing efficiency and urinary oxalate levels was observed and over 60%Hao1 editing efficiency was required to achieve the normalization of urinary oxalate in PH1 rats.These findings suggest that the LNP-mediated base-editing of Hao1c.290-2 A is an efficient and safe approach to PH1 therapy,highlighting its potential utility in clinical settings.展开更多
CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees,yet precise editing remains challenging in these species.Prime editing(PE),a revolutionary technology for ...CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees,yet precise editing remains challenging in these species.Prime editing(PE),a revolutionary technology for precise editing,allows for arbitrary base substitutions and the insertion/deletion of small fragments.In this study,we focused on the model tree poplar 84K(Populus alba 9 P.glandulosa).We used the 2935S promoter to express a fusion protein of spCas9 nickase(nCas9)and engineered Moloney murine leukemia virus(MMLV),and the Arabidopsis thaliana AtU6 promoter to express an engineered PE guide RNA(epegRNA)and Nick gRNA,pioneering the establishment of the Prime Editor 3(PE3)system in dicot poplar.Single-base substitutions,multiple-base substitutions,and small-fragment insertions/deletions were edited into three endogenous target genes.The desired edits were identified in hygromycin-resistant(transformed)calli at seven out of nine target sites,with an average editing efficiency ranging from 0.1 to 3.6%.Furthermore,stable T0 plants contained the desired edits at four out of nine targets,with editing efficiencies ranging from 3.6 to 22.2%.Establishment of the PE3 system provides a powerful tool for the precise modification of the poplar genome.展开更多
Gene-editing technologies have ushered in a significant advancement in plant genetics research and molecular breeding.However,a critical challenge hindering the widespread adoption of these technologies is the efficie...Gene-editing technologies have ushered in a significant advancement in plant genetics research and molecular breeding.However,a critical challenge hindering the widespread adoption of these technologies is the efficient delivery of gene-editing tools.The predominant methods for introducing these tools into plants typically involve Agrobacterium tumefaciens-mediated transformation or particle bombardment(Mao et al.,2019).Unfortunately,these traditional gene delivery methods require delicate and timeconsuming tissue culture procedures and show limited success,especially in medicinal and other less studied plants.A recent breakthrough is the development of the Cut-Dip-Budding(CDB)gene delivery system.The CDB system is highly effective for plants with root-suckering capabilities.It allows the delivery of transgenes and gene-editing tools into plants through hairy root induction followed by shoot regeneration from transformed hairy roots,bypassing the need for tissue culture processes(Cao et al.,2023).展开更多
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 National Key Research and Development Program(2016YFD0500105)the Natural Science Foundation of China(31770191).
文摘Viruses evolve rapidly and continuously threaten animal health and economy,posing a great demand for rapid and efficient genome editing technologies to study virulence mechanism and develop effective vaccine.We present a highly efficient viral genome manipulation method using CRISPR-guided cytidine deaminase.We cloned pseudorabies virus genome into bacterial artificial chromosome,and used CRISPR-guided cytidine deaminase to directly convert cytidine(C)to uridine(U)to induce premature stop mutagenesis in viral genes.The editing efficiencies were 100%.Comprehensive bioinformatic analysis revealed that a large number of editable sites exist in pseudorabies virus(PRV)genomes.Notably,in our study viral genome exists as a plasmid in E.coli,suggesting that this method is virus species-independent.This application of base-editing provided an alternative approach to generate mutant virus and might accelerate study on virulence and vaccine development.
基金supported by the National Natural Science Foundation of China(21922705(to Quanjiang Ji),91753127(to Quanjiang Ji),and 2207783(to Quanjiang Ji))the Shanghai Committee of Science and Technology(19QA1406000(to Quanjiang Ji))+1 种基金the Emergency Key Program of Guangzhou Laboratory(EKPG21-18(to Quanjiang Ji))General Program of Jiangsu Health Committee Foundation(M2020019(to Wei Chen))。
文摘New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable,and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis(M.tuberculosis).Clustered regularly interspaced short palindromic repeat(CRISPR)base editors allow for rapid,robust,and programmed single-base substitutions and gene inactivation,yet no such systems are currently available in M.tuberculosis.By screening distinct CRISPR base editors,we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9(St1Cas9)cytosine base editor(CBE)-but not the widely used Streptococcus pyogenes Cas9(SpCas9)or Lachnospiraceae bacterium Cpf1(LbCpf1)CBEs-is active in mycobacteria.Despite the notable C-to-T conversions,a high proportion of undesired byproducts exists with St1Cas9 CBE.We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor(UGI)or uracil DNA glycosylase(UNG)fusion,yielding two new base editors(CTBE and CGBE)capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis(M.smegmatis).Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif(PAM)sequence for DNA targeting,we engineered a PAM-expanded St1Cas9 variant by means of structureguided protein engineering for the base editors,substantially broadening the targeting scope.We first developed and characterized CTBE and CGBE in M.smegmatis,and then applied CTBE for genome editing in M.tuberculosis.Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics,antibiotic-resistant mechanism study,and drugtarget exploration in M.tuberculosis and related organisms.
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The first editorial board meeting of the World Journal of Hepatology(WJH)was held on November 8,2019 at the Side Bar Grille,Sheraton Boston Hotel,Boston,MA,United States.Ruo-Yu Ma,Director of Editorial Office,on behalf of the Baishideng Publishing Group(BPG),organized the meeting with the great help of Professor Ke-Qin Hu,Journal Editor-in-Chief.There were six editorial board members,including two Editors-in-Chief and one administrative director of the editorial office at the meeting,discussing future strategies of the journal’s development.The editorial board provided BPG a number of suggestions in regard to the business plan and quality control of the WJH.Regarding the business aspect,the editorial board suggested that BPG should advertise the WJH at the international Hepatology and Gastroenterology conferences and promote the WJH via social media.On the scientific aspect,the editorial board suggested that the assessment systems for managing the reviewers and the editorial board members are necessary,and that the BPG should make efforts to attract more high-quality manuscript submissions.An additional comment was to continue to foster a scientific culture for the journal.In conclusion,it was noted that these new ideas expressed during the meeting will bring the WJH to the next level.In the future,the BPG and the editorial board will increase communication and collaboration in order to further the development of the WJH.
文摘Statistical analysis of various data for the papers published in Journal of Shanghai University （ English Edition） from 1997 to 2004 was made. The issues examined include numbers of published papers, disciplinary distribution, constituents of authors, propor- tions of collaborated papers contributed by multiple authors and papers based on cooperation between different institutions, publishing retardation, and citation of references. Suggestions are made for further improving the quality of the Journal.
文摘Acta Geologica Sinica (formerly the Bulletin of the Geological Society of China) beganpublication in 1922, and was published mainly in English through 1952. Since then, thejournal has been published in Chinese with English abstracts. In March 1988, ActaGeologica Sinica appeared in English language translation. The journal is a full translationof the Chinese edition, and follows the Chinese edition by two issues.
文摘Acta Geologica Sinica (formerly the Bulletin of the Geological Society of China) beganpublication in 1922, and was published mainly in English through 1952. Since then, thejournal has been published in Chinese with English abstracts. In March 1988, Acta
文摘Dr.Chunmiao Zheng,a deputy editor-in-chief for Acta Geologica Sinica（English Edition）,received the 2013 O.E.Meinzer Award from the Geological Society of America.Dr.Zheng was also awarded the 2013 M.King Hubbert Award by the National Ground Water Association in the United States.＂It is quite an achievement to receive both of these awards in a lifetime - let alone in the same year!＂ said Professor Mary Anderson of the University of Wisconsin-Madison, a member of the US National Academy of Engineering and Dr. Zheng＇s Ph.D. advisor. Dr. Zheng is currently Chair Professor and Director of the Institute of Water Sciences at Peking University. He has also been the George Lindahl llI Endowed Professor of Hydrogeology at the University of Alabama, United States.
基金supported by the Chuying Scholar Project in Fujian Province and the Innovative Research Project at Fujian Agriculture and Forestry University。
文摘Soybean is the primary source of plant protein for humans.Owing to the indigestibility of the raffinose family of oligosaccharides(RFO),raffinose and stachyose are considered anti-nutritive factors in soybean seeds.Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes,but the carbohydrate profiles invite further modification to lower RFOs.This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis,encoding three raffinose synthases(RS2,RS3,and RS4)and one stachyose synthase.In T1progeny,rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant(rfo-4m)were characterized.The rs2/rs3 mutant showed reduced raffinose and stachyose contents,but the rs4/sts mutant showed only reduced stachyose in seeds.The RFO contents in the rfo-4m mutant were almost eliminated.Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds,including the accumulation of several oligosaccharides-related metabolites.These mutants could contribute to precision breeding of soybean cultivars for soy food production.
基金supported by the National Natural Science Foundation of China(81970324)partially supported by grants from the National Key R&D Program of China 2023YFC3403400。
文摘The innovation of CRISPR/Cas gene editing technology has developed rapidly in recent years.It is widely used in the fields of disease animal model construction,biological breeding,disease diagnosis and screening,gene therapy,cell localization,cell lineage tracking,synthetic biology,information storage,etc.However,developing idealized editors in various fields is still a goal for future development.This article focuses on the development and innovation of non-DSB editors BE and PE in the platform-based CRISPR system.It first explains the application of ideas for improvement such as“substitution”,“combination”,“adaptation”,and“adjustment”in BE and PE development and then catalogues the ingenious inversions and leaps of thought reflected in the innovations made to CRISPR technology.It will then elaborate on the efforts currently being made to develop small editors to solve the problem of AAV overload and summarize the current application status of editors for in vivo gene modification using AAV as a delivery system.Finally,it summarizes the inspiration brought by CRISPR/Cas innovation and assesses future prospects for development of an idealized editor.
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partly funded by the Biological Breeding-Major Projects(Grant No.2023ZD04074 to S.L.)the National Natural Science Foundation of China(Grant No.32188102 to L.X.)+2 种基金National Key Research and Development Program of China(Grant No.2021YFF1000204 to L.X)Hainan Seed Industry Laboratory(Grant No.B23CJ0208 to L.X.)the National Engineering Research Centre of Crop Molecular Breeding,and the National Natural Science Foundation of China(Grant No.32100328)。
文摘The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement.CRISPR/Cas12i3,which belongs to the ClassⅡTypeⅤ-ⅡCas system,has attracted extensive attention recently due to its smaller protein size and less restricted canonical"TTN"protospacer adjacent motif(PAM).However,due to its relatively lower editing efficiency,Cas12i3-mediated multiplex gene editing has not yet been documented in plants.Here,we fused four 5'exonucleases(Exo)including T5E,UL12,PapE,ME15 to the N terminal of an optimized Cas12i3 variant(Cas12i3-5M),respectively,and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines.We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M,respectively.Notably,the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61%across the six tested endogenous genes.We further demonstrated that,although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing,UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three,four,five or six genes with efficiencies of 82.76%,61.36%,52.94%,and 51.06%in rice stable lines,respectively.Together,we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice.The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.
基金support from National Natural Science Foundation of China(82302497).
文摘CGBE(C-to-G base editor)systems,pivotal components within the base editing arsenal,enable the precise conversion of cytosines to guanines.However,conventional cytidine deaminases possess non-specific single-stranded DNA binding properties,leading to off-target effects and safety concerns.The Cas-embedding strategy,which involves embedding functional proteins like deaminases within the Cas9 enzyme’s architecture,emerges as a method to mitigate these off-target effects.Our study pioneers the application of the Cas-embedding strategy to CGBE systems,engineering a suite of novel CGBE editors,CE-CGBE.The CE-CGBE that incorporated eA3A,RBMX and Udgx excelled in editing efficiency,editing purity,and indel formation was named HF-CGBE.HF-CGBE showed no significant difference in off-target effects compared to the negative control group for both DNA and RNA.In summary,the novel HF-CGBE editors we propose expand the base editing toolbox and provide therapeutic approaches for related pathogenic mutations.
基金supported by the STI 2030-Major Projects(2023ZD04074)the National Key Research and Development Program of China(2023YFD1202900)+2 种基金the Nanfan special project of the Chinese Academy of Agricultural Sciences(YBXM2313)the Hainan Seed Industry Laboratory(project of B23CJ0208)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.
基金supported by the Ministry of Agriculture and Rural Affairs of China,the Jiangsu Province Natural Science Foundation(grant nos.BK20212010 and BK20230038)the Jiangsu Province Key Research and Development Program(grant no.BE2022383)+4 种基金the Hainan Seed Industry Laboratory(grant no.B21HJ1004)the Zhongshan Biological Breeding Laboratory(grant no.ZSBBL-KY2023-04)the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University(grant no.NAUSY-ZZ01)the Bingtuan Key Science and Technology Program of Xinjiang Province(grant no.2023AB006-02)the National Natural Science Foundation of China(grant no.31872806).
文摘The programmable CRISPR-Cas genome editing technology,adopted from prokaryotic adaptive immune systems,has revolutionized genome engineering in plants(Liu et al.,2022a).Many efforts have been made to improve the activity,specificity,and protospacer adjacent motif(PAM)variants of Class 2 Cas nucleases,such as Cas9,Cas12a,and Cas12b(Liu et al.,2022a).However,their large size(∼1000–1400 amino acids)poses a challenge in scenarios requiring a compact Cas nuclease,particularly in urgent situations like plant virus-induced genome editing(Cheuk and Houde,2018;Li et al.,2021;Varanda et al.,2021).
文摘Ongoing advances in genomics and genetics have led researchers to highlight the significant role of large DNA structural variants(SVs)in genetic diversity.SVs are genomic sequence variants of various types,including insertions,deletions,inversions,translocations,and copy number variants(CNVs or duplications).These SVs,which are usually of 1 kb or more,can have profound impacts at both the individual and species level.They are also often linked to the initiation and progression of complex genetic diseases(Stankiewicz and Lupski,2010).
基金partially supported by the National Natural Science Foundation of China (82470794,32025023 and 32230064)the National Key Research and Development Program of China (2023YFC3403400 to Li,D)a grant from the Science and Technology Commission of Shanghai Municipality (22YF1426900)。
文摘Primary hyperoxaluria type 1(PH1)is a severe hereditary disease,leading to the accumulation of oxalate in multiple organs,particularly the kidney.Hydroxyacid oxidase 1(HAO1),a pivotal gene involved in oxalate production,is an approved target for the treatment of PH1.In this study,we demonstrated the discovery of several novel therapeutic sites of the Hao1 gene and the efficient editing of Hao1c.290-2 A in vivo with lipid nanoparticles(LNP)delivered adenine base editing(ABE)mRNA.A single infusion of LNP-ABE resulted in a near-complete knockout of Hao1 in the liver,leading to the sustainable normalization of urinary oxalate(for at least 6 months)and complete rescue of the pathophysiology in PH1 rats.Additionally,a significant correlation between Hao1 editing efficiency and urinary oxalate levels was observed and over 60%Hao1 editing efficiency was required to achieve the normalization of urinary oxalate in PH1 rats.These findings suggest that the LNP-mediated base-editing of Hao1c.290-2 A is an efficient and safe approach to PH1 therapy,highlighting its potential utility in clinical settings.
基金supported by the National Key Research and Development Program of China(2021YFD2200201)the China National Rice Research Institute Key Research and Development Project(CNRRI-2020-01)the Central Public-interest Scientific Institution Basal Research Fund,China(CPSIBRF-CNRRI-202203).
文摘CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees,yet precise editing remains challenging in these species.Prime editing(PE),a revolutionary technology for precise editing,allows for arbitrary base substitutions and the insertion/deletion of small fragments.In this study,we focused on the model tree poplar 84K(Populus alba 9 P.glandulosa).We used the 2935S promoter to express a fusion protein of spCas9 nickase(nCas9)and engineered Moloney murine leukemia virus(MMLV),and the Arabidopsis thaliana AtU6 promoter to express an engineered PE guide RNA(epegRNA)and Nick gRNA,pioneering the establishment of the Prime Editor 3(PE3)system in dicot poplar.Single-base substitutions,multiple-base substitutions,and small-fragment insertions/deletions were edited into three endogenous target genes.The desired edits were identified in hygromycin-resistant(transformed)calli at seven out of nine target sites,with an average editing efficiency ranging from 0.1 to 3.6%.Furthermore,stable T0 plants contained the desired edits at four out of nine targets,with editing efficiencies ranging from 3.6 to 22.2%.Establishment of the PE3 system provides a powerful tool for the precise modification of the poplar genome.
基金supported by the National Key R&D Program of China (2021YFA1300404 to J.-K.Z)National Natural Science Foundation of China (32188102 to J.-K.Z)by Bellagen Biotechnology Co. Ltd., Jinan, China
文摘Gene-editing technologies have ushered in a significant advancement in plant genetics research and molecular breeding.However,a critical challenge hindering the widespread adoption of these technologies is the efficient delivery of gene-editing tools.The predominant methods for introducing these tools into plants typically involve Agrobacterium tumefaciens-mediated transformation or particle bombardment(Mao et al.,2019).Unfortunately,these traditional gene delivery methods require delicate and timeconsuming tissue culture procedures and show limited success,especially in medicinal and other less studied plants.A recent breakthrough is the development of the Cut-Dip-Budding(CDB)gene delivery system.The CDB system is highly effective for plants with root-suckering capabilities.It allows the delivery of transgenes and gene-editing tools into plants through hairy root induction followed by shoot regeneration from transformed hairy roots,bypassing the need for tissue culture processes(Cao et al.,2023).
