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
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 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
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
Xenotransplantation has entered the clinical phase in an effort to address the global organ shortage.However,recent clinical studies have revealed that current xenografts from gene-edited(GE)pigs still pose a risk of ...Xenotransplantation has entered the clinical phase in an effort to address the global organ shortage.However,recent clinical studies have revealed that current xenografts from gene-edited(GE)pigs still pose a risk of immune rejection and biosafety concerns.In this study,we successfully produced a large batch of 582 GE cloned(GEC)pigs with 10-(GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hTBM/hCD39/hEPCR/hCD47)gene edits via gene editing and somatic cell cloning technologies,and successfully obtained the F1 generation.Phenotypic character ization of 10-GEC pigs revealed the deletion of three xenoantigens and the expression of seven human transgenes across various tissues.Digital droplet polymerase chain reaction and whole-genome sequencing revealed two cop ies of hCD46/hCD55/hCD59/hTBM/hCD39 and one copy of hEPCR/hCD47 in the pig genome with minimal off-target effects or damage to the porcine functional genes.The validation results showed that 10-GEC pigs could effectively inhibit attacks from human antibodies,complement and macrophages on porcine endothelial cells,and alleviated coagulation abnormalities between pigs and humans.Large-scale screening of pathogens revealed no evidence of 47 pathogens,including cytomegalovirus,in our 10-GEC pigs.Kidney,heart and liver xenografts from these 10-GEC pigs were transplanted into nonhuman primates(NHPs),which worked normally without hyperacute rejection(HAR).Among NHPs,the heart and liver orthotopic transplant recipients survived for 3 and 4 days,respectively,while the two kidney transplant recipients survived for 23 and 16 days,respectively.Pathological analysis showed interstitial hemorrhage and fibrosis,cellular hyperplasia with minor antibodies and complement deposition,but significantly reduced infiltration of CD68^(+)macrophages in 10-GEC pig kidney xenografts.In summary,we success fully produced specific pathogen-free 10-GEC donor pigs that resulted in effective mitigation of immune rejection upon multiorgan transplantation to NHPs.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
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
Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance ...Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.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
Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regu...Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.展开更多
The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats(CRISPR) and programmable nucleases to make precise modifications to geneti...The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats(CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.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.
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 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.
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Major Science and Technology Project of Yunnan Province(202102AA100054,202102AA310047)Basic Strengthening Program(2023-JCJQ-ZD-118-00/173)+2 种基金Xingdian Talent Support Program(XDYC-KJLJ-2022-0004)Major Science and Technology Project,Yunnan Province(202102AA310047,202102AA100054)National Key R&D Program of China(Grant No.2019YFA0110700).
文摘Xenotransplantation has entered the clinical phase in an effort to address the global organ shortage.However,recent clinical studies have revealed that current xenografts from gene-edited(GE)pigs still pose a risk of immune rejection and biosafety concerns.In this study,we successfully produced a large batch of 582 GE cloned(GEC)pigs with 10-(GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hTBM/hCD39/hEPCR/hCD47)gene edits via gene editing and somatic cell cloning technologies,and successfully obtained the F1 generation.Phenotypic character ization of 10-GEC pigs revealed the deletion of three xenoantigens and the expression of seven human transgenes across various tissues.Digital droplet polymerase chain reaction and whole-genome sequencing revealed two cop ies of hCD46/hCD55/hCD59/hTBM/hCD39 and one copy of hEPCR/hCD47 in the pig genome with minimal off-target effects or damage to the porcine functional genes.The validation results showed that 10-GEC pigs could effectively inhibit attacks from human antibodies,complement and macrophages on porcine endothelial cells,and alleviated coagulation abnormalities between pigs and humans.Large-scale screening of pathogens revealed no evidence of 47 pathogens,including cytomegalovirus,in our 10-GEC pigs.Kidney,heart and liver xenografts from these 10-GEC pigs were transplanted into nonhuman primates(NHPs),which worked normally without hyperacute rejection(HAR).Among NHPs,the heart and liver orthotopic transplant recipients survived for 3 and 4 days,respectively,while the two kidney transplant recipients survived for 23 and 16 days,respectively.Pathological analysis showed interstitial hemorrhage and fibrosis,cellular hyperplasia with minor antibodies and complement deposition,but significantly reduced infiltration of CD68^(+)macrophages in 10-GEC pig kidney xenografts.In summary,we success fully produced specific pathogen-free 10-GEC donor pigs that resulted in effective mitigation of immune rejection upon multiorgan transplantation to NHPs.
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 grants from the National Natural Science Foundation of China(31788103 and 31971370)the National Transgenic Science and Technology Program(2016ZX08010002 and 2018ZX0801002B)。
文摘Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.
基金supported by the STI 2030-Major Projects(2023ZD04074)the National Key Research and Development Program of China(2023YFD1202900)+2 种基金the Nanfan special project of the Chinese Academy of Agricultural Sciences(YBXM2313)the Hainan Seed Industry Laboratory(project of B23CJ0208)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.
基金supported by grants from the National Key Research and Development Program of China (2016YFD0101804)the National Natural Science Foundation of China (31788103 and 31420103912)as well as the Chinese Academy of Sciences (QYZDY-SSWSMC030 and GJHZ1602)
文摘Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.
基金supported by the National Natural Science Foundation of China (81830004, 31922046, 31770057, 31722036, 31930016, 31870893)the Sanming Project of Medicine in Shenzhen (SZSM202011017)+5 种基金the National Key Research and Development Program of China (2018YFA0801401, 2019YFA0110800, 2018YFA0107703, 2019YFA0110000, 2020YFA0707800, 2020YFA0707600)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030403, XDA16010503)Beijing Municipal Science & Technology Commission (Z181100001318009)Beijing Advanced Innovation Center for Genomics at Peking Universitythe Peking-Tsinghua Center for Life Sciencesthe National Major Science & Technology Project for Control and Prevention of Major Infectious Diseases in China (2018ZX10301401)
文摘The steady progress in genome editing, especially genome editing based on the use of clustered regularly interspaced short palindromic repeats(CRISPR) and programmable nucleases to make precise modifications to genetic material, has provided enormous opportunities to advance biomedical research and promote human health. The application of these technologies in basic biomedical research has yielded significant advances in identifying and studying key molecular targets relevant to human diseases and their treatment. The clinical translation of genome editing techniques offers unprecedented biomedical engineering capabilities in the diagnosis, prevention, and treatment of disease or disability. Here, we provide a general summary of emerging biomedical applications of genome editing, including open challenges. We also summarize the tools of genome editing and the insights derived from their applications, hoping to accelerate new discoveries and therapies in biomedicine.
