Bacterial blight(BB),caused by Xanthomonas oryzae pathovar oryzae(Xoo),poses a significant threat to rice production,particularly in Asia and West Africa.Breeding resistance against BB in elite rice varieties is cruci...Bacterial blight(BB),caused by Xanthomonas oryzae pathovar oryzae(Xoo),poses a significant threat to rice production,particularly in Asia and West Africa.Breeding resistance against BB in elite rice varieties is crucial to advancing rice breeding program and supporting smallholder farmers.Transcription Activator-Like effectors(TALes)are key virulence factors in Xoo,with some targeting the susceptibility(S)genes such as the sugar transporter SWEET genes in rice.Among these,SWEET14 is an important S gene,with its promoter bound by the TALe TalC which exists across all sequenced African Xoo isolates.In the present study,we utilized CRISPR/Cas9-based cytidine and adenine base editors to alter the effector binding element(EBE)of TalC in the promoter of SWEET14 in rice cultivars Kitaake,IR24,and Zhonghua 11.Mutations with C to T changes in EBE led to reduced SWEET14 induction by TalC-containing Xoo strains,resulting in resistance to African Xoo isolates reliant on TalC for virulence.Conversely,A to G changes retained SWEET14 inducibility and susceptibility to Xoo in edited lines.Importantly,no off-target mutations were detected at predicted sites,and the edited lines exhibited no obvious defects in major agronomic traits in Kitaake.These results underscore the effectiveness of base editing systems for both molecular biology research and crop improvement endeavors.展开更多
Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present...Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present the development and evaluation of a novel glycosylase-based cytosine base editor(gCBE)for efficient C-to-G editing in rice.Unlike traditional cytosine base editors,which rely on cytosine deamination,gCBE directly excises cytosine to generate an apurinic/apyrimidinic(AP)site,thus circumventing the deamination step and reducing the production of C-to-T byproducts.We constructed several gCBE variants,including N-gCBE,M-gCBE,and C-gCBE,by fusing engineered human UDG2(UNG*)to SpCas9 nickase(nSpCas9,D10A)and tested their editing efficiency and specificity in rice.Our results demonstrate that M-gCBE achieved efficient C-to-G editing(6.3%to 37.5%)similar to OsCGBE(9.4%to 28.1%)at most targets,though with site-dependent variations.Notably,gCBE tools showed a marked reduction in C-to-T byproducts,with average C-to-T mutation rates of 12.5%for N-gCBE and 16.7%for M-gCBE,compared to 53.1%for OsCGBE.Notably,both N-gCBE and M-gCBE were capable of generating homozygous C-to-G mutations in the T_(0)generation,a key advantage over OsCGBE,which predominantly generated C-to-T mutations.Off-target analysis revealed minimal off-target effects with M-gCBE,highlighting its potential for high-precision genome editing.These findings suggest that gCBE tools,particularly M-gCBE,are highly efficient and precise,providing an advanced solution for C-to-G editing in plants and offering promising applications for crop improvement.展开更多
The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9), a ...The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing(e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.展开更多
Genome editing technologies have revolutionized the field of plant science by enabling targeted modification of plant genomes and are emerging as powerful tools for both plant gene functional analyses and crop improve...Genome editing technologies have revolutionized the field of plant science by enabling targeted modification of plant genomes and are emerging as powerful tools for both plant gene functional analyses and crop improvement. Although homology-directed repair(HDR)is a feasible approach to achieve precise gene replacement and base substitution in some plant species, the dominance of the non-homologous end joining pathway and low efficiency of HDR in plant cells have limited its application. Base editing has emerged as an alternative tool to HDR-mediated replacement, facilitating precise editing of plant genome by converting one single base to another in a programmable manner without a doublestranded break and a donor repair template. In this review, we summarize the latest developments in base-editing technologies as well as their underlying mechanisms. We review current applications of these technologies in plant species. Finally, we address the challenges and future perspectives of this emerging technology in plants.展开更多
Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutatio...Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.展开更多
Base editing, as an expanded clustered regularly interspaced short palindromic repeats(CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9...Base editing, as an expanded clustered regularly interspaced short palindromic repeats(CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9 ortholog from Staphylococcus aureus(SaCas9), is important in genome editing because it can edit sites with HHHAAT protospacer adjacent motif(PAM) that the canonical Streptococcus pyogenes Cas9(SpCas9) or its variants(e.g. xCas9, Cas9-NG) cannot. However, several technical parameters of SaKKH involved base editors have not been well defined and this uncertainty limits their application. We developed an effective multiplex cytosine base editor(SaKKHn-pBE) and showed that it recognized NNARRT, NNCRRT, NNGRGT, and NNTRGT PAMs. Based on 27 targets tested, we defined technical parameters of SaKKHn-pBE including the editing window, the preferred sequence context, and the mutation type. The editing efficiency was further improved by modification of the SaKKH sgRNA. These advances can be applied in future research and molecular breeding in rice and other plants.展开更多
Genome editing has undergone rapid development in recent years,yielding new approaches to make precise changes in genes.In this review,we discuss the development of various adenine and cytosine base-editing technologi...Genome editing has undergone rapid development in recent years,yielding new approaches to make precise changes in genes.In this review,we discuss the development of various adenine and cytosine base-editing technologies,which share the ability to make specific base changes at specific sites in the genome.We also describe multiple applications of base editing in vitro and in vivo.Finally,as a practical example,we demonstrate the use of a cytosine base editor and an adenine base editor in human cells to introduce and then correct a prevalent mutation responsible for hereditary tyrosinemia type 1.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
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
Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were f...Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.展开更多
Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding....Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.展开更多
Dear Editor The newly developed CRISPR/Cas9-mediated base editing technology with cytosine deaminase is capable of precisely and efficiently introducing point mutations at the target genomic locus, which does not requ...Dear Editor The newly developed CRISPR/Cas9-mediated base editing technology with cytosine deaminase is capable of precisely and efficiently introducing point mutations at the target genomic locus, which does not require double-stranded DNA breaks or any donor templates and thus exhibit a great potential for gene correction and genetic diversification in yeasts, plants, and mammalian and human cells （Komor et al., 2016; Nishida et al., 2016; Lu and Zhu, 2017; Ren et al., 2017）.展开更多
Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing...Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strategies have been attempted to optimize both base editor(BE)and prime editor(PE)in order to improve the precise editing efficacy,specificity,and expand the targeting scopes.Here,we summarize the latest development of various BEs and PEs,as well as their applications in plants.Based on these progresses,we recommend the appropriate BEs and PEs for both basic plant research and crop improvement.Moreover,we propose the perspectives for further optimization of these two editors.We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.展开更多
Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the ac...Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.展开更多
Many human genetic diseases,including Hutchinson-Gilford progeria syndrome(HGPS),are caused by single point mutations.HGPS is a rare disorder that causes premature aging and is usually caused by a de novo point mutati...Many human genetic diseases,including Hutchinson-Gilford progeria syndrome(HGPS),are caused by single point mutations.HGPS is a rare disorder that causes premature aging and is usually caused by a de novo point mutation in the LMNA gene.Base editors(BEs)composed of a cytidine deaminase fused to CRISPR/Cas9 nickase are highly efficient at inducing C to T base conversions in a programmable manner and can be used to generate animal disease models with single amino-acid substitutions.Here,we generated the first HGPS monkey model by delivering a BE mRNA and guide RNA(gRNA)targeting the LMNA gene via microinjection into monkey zygotes.Five out of six newborn monkeys carried the mutation specifically at the target site.HGPS monkeys expressed the toxic form of lamin A,progerin,and recapitulated the typical HGPS phenotypes including growth retardation,bone alterations,and vascular abnormalities.Thus,this monkey model genetically and clinically mimics HGPS in humans,demonstrating that the BE system can efficiently and accurately generate patient-specific disease models in non-human primates.展开更多
The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) ...The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing.展开更多
Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes.Therefore, it takes a lon...Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes.Therefore, it takes a long time to get homozygous mutants. Recently developed base editing(BE) system,which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons(i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sg RNA targeting CD160 and a mixture of sg RNAs targeting VISTA and CD160,respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.展开更多
The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats(CRISPR)-bas...The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats(CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein(Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases(CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.展开更多
Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricult...Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.展开更多
Pigs are one of the most important domesticated animals and have great value in agriculture and biomedicine.Single nucleotide polymorphisms(SNPs)are a dominant type of genetic variation among individual pigs and contr...Pigs are one of the most important domesticated animals and have great value in agriculture and biomedicine.Single nucleotide polymorphisms(SNPs)are a dominant type of genetic variation among individual pigs and contribute to the formation of traits.Precision single base substitution provides a strategy for accurate genetic improvement in pig production with the characterization of functional SNPs and genetic variants in pigs.Base editing has recently been developed as the latest gene-editing tool that can directly make changes in single nucleotides without introducing double-stranded DNA breaks(DSBs),providing a promising solution for precise genetic modification in large animals.This review summarizes gene-editing developments and highlights recent genetic dissection related to SNPs in major economic traits which may have the potential to be modified using SNP-editing applications.In addition,limitations and future directions of base editing in pig breeding are discussed.展开更多
基金supported by a sub-award to the University of Missouri from the Heinrich Heine University of Dusseldorf funded by the Bill&Melinda Gates Foundation(OPP1155704)(Bing Yang)and the China Scholar Council(Chenhao Li,as a joint Ph.D.student).
文摘Bacterial blight(BB),caused by Xanthomonas oryzae pathovar oryzae(Xoo),poses a significant threat to rice production,particularly in Asia and West Africa.Breeding resistance against BB in elite rice varieties is crucial to advancing rice breeding program and supporting smallholder farmers.Transcription Activator-Like effectors(TALes)are key virulence factors in Xoo,with some targeting the susceptibility(S)genes such as the sugar transporter SWEET genes in rice.Among these,SWEET14 is an important S gene,with its promoter bound by the TALe TalC which exists across all sequenced African Xoo isolates.In the present study,we utilized CRISPR/Cas9-based cytidine and adenine base editors to alter the effector binding element(EBE)of TalC in the promoter of SWEET14 in rice cultivars Kitaake,IR24,and Zhonghua 11.Mutations with C to T changes in EBE led to reduced SWEET14 induction by TalC-containing Xoo strains,resulting in resistance to African Xoo isolates reliant on TalC for virulence.Conversely,A to G changes retained SWEET14 inducibility and susceptibility to Xoo in edited lines.Importantly,no off-target mutations were detected at predicted sites,and the edited lines exhibited no obvious defects in major agronomic traits in Kitaake.These results underscore the effectiveness of base editing systems for both molecular biology research and crop improvement endeavors.
基金supported by the National Natural Science Foundation of China(82404798)the Natural Science Foundation of Sichuan Province(2024NSFSC1831)+1 种基金the National Key Laboratory for Tropical Crop Breeding(NKLTCB-RC202403,NKLTCBZRJJ4)the Hainan Seed Industrial Laboratory(B22C1000P).
文摘Base editors are essential tools for precise genome editing in plants.However,achieving high efficiency in C-to-G editing while minimizing byproduct and offtarget mutations remains challenging.In this study,we present the development and evaluation of a novel glycosylase-based cytosine base editor(gCBE)for efficient C-to-G editing in rice.Unlike traditional cytosine base editors,which rely on cytosine deamination,gCBE directly excises cytosine to generate an apurinic/apyrimidinic(AP)site,thus circumventing the deamination step and reducing the production of C-to-T byproducts.We constructed several gCBE variants,including N-gCBE,M-gCBE,and C-gCBE,by fusing engineered human UDG2(UNG*)to SpCas9 nickase(nSpCas9,D10A)and tested their editing efficiency and specificity in rice.Our results demonstrate that M-gCBE achieved efficient C-to-G editing(6.3%to 37.5%)similar to OsCGBE(9.4%to 28.1%)at most targets,though with site-dependent variations.Notably,gCBE tools showed a marked reduction in C-to-T byproducts,with average C-to-T mutation rates of 12.5%for N-gCBE and 16.7%for M-gCBE,compared to 53.1%for OsCGBE.Notably,both N-gCBE and M-gCBE were capable of generating homozygous C-to-G mutations in the T_(0)generation,a key advantage over OsCGBE,which predominantly generated C-to-T mutations.Off-target analysis revealed minimal off-target effects with M-gCBE,highlighting its potential for high-precision genome editing.These findings suggest that gCBE tools,particularly M-gCBE,are highly efficient and precise,providing an advanced solution for C-to-G editing in plants and offering promising applications for crop improvement.
基金financially supported by the National Natural Science Foundation of China (32000454)Provincial Natural Science Foundation of Hebei for Excellent Young Scholar (C2020204062)+1 种基金Program for Young Talents of Hebei Education Department (BJ2021025)Starting Grant from Hebei Agricultural University (YJ201958)。
文摘The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing(e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.
基金partly funded by the Transgenesis Initiative Project supported by the Ministry of Agriculture and Rural Affairs of China(2019ZX08010001,2019ZX08010003)the Central Non-Profit Fundamental Research Funding supported by the Institute of Crop Sciences,Chinese Academy of Agricultural Sciences(S2018QY05)。
文摘Genome editing technologies have revolutionized the field of plant science by enabling targeted modification of plant genomes and are emerging as powerful tools for both plant gene functional analyses and crop improvement. Although homology-directed repair(HDR)is a feasible approach to achieve precise gene replacement and base substitution in some plant species, the dominance of the non-homologous end joining pathway and low efficiency of HDR in plant cells have limited its application. Base editing has emerged as an alternative tool to HDR-mediated replacement, facilitating precise editing of plant genome by converting one single base to another in a programmable manner without a doublestranded break and a donor repair template. In this review, we summarize the latest developments in base-editing technologies as well as their underlying mechanisms. We review current applications of these technologies in plant species. Finally, we address the challenges and future perspectives of this emerging technology in plants.
基金supported by the Key Area Research and Development Program of Guangdong Province(2018B020202008)the National Natural Science Foundation of China(31771808)+2 种基金Beijing Municipal Science and Technology Project(D171100007717001)the National Key Research and Development Program of China(2016YFD0101803)National Engineering Laboratory for Crop Molecular Breeding。
文摘Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.
基金supported by the Beijing Scholars Program[BSP041]。
文摘Base editing, as an expanded clustered regularly interspaced short palindromic repeats(CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9 ortholog from Staphylococcus aureus(SaCas9), is important in genome editing because it can edit sites with HHHAAT protospacer adjacent motif(PAM) that the canonical Streptococcus pyogenes Cas9(SpCas9) or its variants(e.g. xCas9, Cas9-NG) cannot. However, several technical parameters of SaKKH involved base editors have not been well defined and this uncertainty limits their application. We developed an effective multiplex cytosine base editor(SaKKHn-pBE) and showed that it recognized NNARRT, NNCRRT, NNGRGT, and NNTRGT PAMs. Based on 27 targets tested, we defined technical parameters of SaKKHn-pBE including the editing window, the preferred sequence context, and the mutation type. The editing efficiency was further improved by modification of the SaKKH sgRNA. These advances can be applied in future research and molecular breeding in rice and other plants.
基金This work was supported by U.S.National Institutes of Health grant R35-HL145203 and the Winkelman Family Fund in Cardiovascular Innovation(K.M.).K.M.is a co-founder and advisor of Verve Therapeutics and an advisor of Variant Bio.
文摘Genome editing has undergone rapid development in recent years,yielding new approaches to make precise changes in genes.In this review,we discuss the development of various adenine and cytosine base-editing technologies,which share the ability to make specific base changes at specific sites in the genome.We also describe multiple applications of base editing in vitro and in vivo.Finally,as a practical example,we demonstrate the use of a cytosine base editor and an adenine base editor in human cells to introduce and then correct a prevalent mutation responsible for hereditary tyrosinemia type 1.
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 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supported by funding from the National Natural Science Foundation of China(31801016)the Agricultural Variety Improvement Project of Shandong Province(2019LZGC015)。
文摘Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.
基金supported by grants from the National Transgenic Science and Technology Program of China(2019ZX08010-003)to F.Y.the National Natural Science Foundation of China(31871948)the Fundamental Research Funds,and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(Y2020PT26)to H.Z.
文摘Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.
基金This study was supported by grants from the National Key Research and Development Program of China （2017YFD0200900） and the Agricultural Science and Technology Innovation Program of The Chinese Academy of Agricultural Sciences to H.Z., and a grant from the National Natural Science Foundation of China （31701780） to F.Y.
文摘Dear Editor The newly developed CRISPR/Cas9-mediated base editing technology with cytosine deaminase is capable of precisely and efficiently introducing point mutations at the target genomic locus, which does not require double-stranded DNA breaks or any donor templates and thus exhibit a great potential for gene correction and genetic diversification in yeasts, plants, and mammalian and human cells （Komor et al., 2016; Nishida et al., 2016; Lu and Zhu, 2017; Ren et al., 2017）.
基金funded by National Key Research and Development Program of China(2021YFF1000204)Nanfan Special Project,CAAS(ZDXM03)+1 种基金Hainan Yazhou Bay Seed Lab(B21HJ0215,B21Y10205)Key Laboratory of Gene Editing Technologies(Hainan),Ministry of Agriculture and Rural Affairs,China。
文摘Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strategies have been attempted to optimize both base editor(BE)and prime editor(PE)in order to improve the precise editing efficacy,specificity,and expand the targeting scopes.Here,we summarize the latest development of various BEs and PEs,as well as their applications in plants.Based on these progresses,we recommend the appropriate BEs and PEs for both basic plant research and crop improvement.Moreover,we propose the perspectives for further optimization of these two editors.We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.
基金supported by grants from the National Key R&D Program of China(2018YFA0900600)the National Natural Science Foundation of China(31900301,31872933 and 31971370)the Chinese Academy of Sciences(QYZDY-SSW-SMC030).
文摘Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.
基金We are grate to Xinglong Chen,Ziyi Zhao,Baohong Tian and all members from animal facility of the Yunnan Key Laboratory of Primate Biomedical Research for excellent animal welfare and husbandry.We thank Jing He for her technical assistance.The author would like to thank Gabriella Rudy for constructive criticism of the manuscript.This work was supported by the National Key Research and Development Program(2016YFA0101401)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16010100)+2 种基金the National Key Research and Development Program(2018YFA0801403,2018YFC2000100)the National Natural Science Foundation of China(Grant Nos.81921006,81625009,91749202,91949209,81822018,91749123,81671377)Youth Innovation Promotion Association of CAS(2016093).
文摘Many human genetic diseases,including Hutchinson-Gilford progeria syndrome(HGPS),are caused by single point mutations.HGPS is a rare disorder that causes premature aging and is usually caused by a de novo point mutation in the LMNA gene.Base editors(BEs)composed of a cytidine deaminase fused to CRISPR/Cas9 nickase are highly efficient at inducing C to T base conversions in a programmable manner and can be used to generate animal disease models with single amino-acid substitutions.Here,we generated the first HGPS monkey model by delivering a BE mRNA and guide RNA(gRNA)targeting the LMNA gene via microinjection into monkey zygotes.Five out of six newborn monkeys carried the mutation specifically at the target site.HGPS monkeys expressed the toxic form of lamin A,progerin,and recapitulated the typical HGPS phenotypes including growth retardation,bone alterations,and vascular abnormalities.Thus,this monkey model genetically and clinically mimics HGPS in humans,demonstrating that the BE system can efficiently and accurately generate patient-specific disease models in non-human primates.
基金funded by the Genetically Modified Breeding Major Project(2016ZX08010-002-008)the National Natural Science Foundation of China(31701405)the Natural Science Foundation of Anhui Province,China(1708085QC60)。
文摘The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3(BE3) and targeted activation-induced cytidine deaminase(CDA)(target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase(UDG) inhibitor(UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a highfidelity and high-efficiency solution for rice genomic base editing.
基金supported by the National Key R&D Program(2016YFC0905901 to X.H.,2016YFA0503300 to X.G.)the NSFC(81771641 to X.G.)+1 种基金Fok Ying Tung Education Foundation(161037 to X.G.)Local Grants(17JC1420103 to X.H.,SKLRM-K201502 to X.G.)
文摘Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes.Therefore, it takes a long time to get homozygous mutants. Recently developed base editing(BE) system,which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons(i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sg RNA targeting CD160 and a mixture of sg RNAs targeting VISTA and CD160,respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.
基金supported by the National Natural Science Foundation of China (82270355, 82270354, 81970134, 82030011, 31630093)the National Key Research and Development Program of China (2019YFA0801601, 2021YFA1101801)。
文摘The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats(CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein(Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases(CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.
基金supported by grants from the Shenzhen Science and Technology Program(KQTD20180411143628272)Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District(PT202101-02)+3 种基金the Hainan Yazhou Bay Seed Lab(B21HJ0215),the National Natural Science Foundation of China(32102294)the China National Postdoctoral Program for Innovative Talents(BX2020378)the China Postdoctoral Science Foundation(2020M672902)the Central Publicinterest Scientific Institution Basal Research Fund(Y2022PT24).
文摘Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.
基金supported by the National Natural Science Foundation of China(81671274,31925036,31272440,and 31801031)the National Transgenic Project of China(2016ZX08009003006-007)the Elite Youth Program of the Chinese Academy of Agricultural Sciences(ASTIP-IAS05)。
文摘Pigs are one of the most important domesticated animals and have great value in agriculture and biomedicine.Single nucleotide polymorphisms(SNPs)are a dominant type of genetic variation among individual pigs and contribute to the formation of traits.Precision single base substitution provides a strategy for accurate genetic improvement in pig production with the characterization of functional SNPs and genetic variants in pigs.Base editing has recently been developed as the latest gene-editing tool that can directly make changes in single nucleotides without introducing double-stranded DNA breaks(DSBs),providing a promising solution for precise genetic modification in large animals.This review summarizes gene-editing developments and highlights recent genetic dissection related to SNPs in major economic traits which may have the potential to be modified using SNP-editing applications.In addition,limitations and future directions of base editing in pig breeding are discussed.
