Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from S...Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from South/Southeast Asian wild O. nivara(Jing et al., 2023). Genes like An-1, BH4, PROG1,SH4, Rc, Rd, and GS3—which regulate awn length, hull color,til er angle, seed shattering, pericarp color, seed length, and thousand-grain weight, respectively—were selected against during domestication to form modern O. sativa(Yu et al., 2021).However, domestication and yield-focused breeding eliminated wild rice's valuable genes(e.g., for disease resistance, stress tolerance, nutrition), narrowing genetic diversity and impeding efforts to meet growing societal demands.展开更多
Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using ...Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.展开更多
Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on...Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.展开更多
The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of t...The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.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
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
CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(M...CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(Medicago sativa L.),the most important forage legume worldwide,remains a formidable challenge.Here,we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves.We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system.Subsequently,we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA(PTG)expression cassettes,each consisting of three tRNA-sgRNA units,to simultaneously edit three or four alfalfa genes,coupled with the visual reporter RH1 or RUBY.This toolkit showed efficient multiplex editing in the hairy root system with visual selection.We successfully obtained regenerated,red-colored shoots resulting from the stable transformation of alfalfa.These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa.Our improved CRISPR/Cas9 multiplex system enables convenient,high-efficiency multiplex genome editing in alfalfa,providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.展开更多
Dear Editor,Receptor-like kinases(RLKs)constitute a large superfamily that regulates diverse biological processes in plants(De Smet et al.,2009).In the perennial tree Populus,nearly 200 RLKs are highly expressed in de...Dear Editor,Receptor-like kinases(RLKs)constitute a large superfamily that regulates diverse biological processes in plants(De Smet et al.,2009).In the perennial tree Populus,nearly 200 RLKs are highly expressed in developing xylem,suggesting important roles in wood formation(Song et al.,2011;Zan et al.,2013;Xie et al.,2023).展开更多
CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination(HR) ability. Her...CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination(HR) ability. Here, a deaminase-assisted base editor d Cas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9(dCas9), the cytidine deaminase from Petromyzon marinus(PmCDA1), the uracil DNA glycosylase inhibitor(UGI) and the protein degradation tag(LVA tag). Using d Cas9-CDA-ULstr , we achieved single-, double-and triple-point mutations(cytosine-to-thymine substitutions)at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes(with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the d Cas9-CDA-ULstr -based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.展开更多
The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V ...The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl （also known as Cas12a） system is a two-component transcriptional unit （TCTU） in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase （pol） II promoter, whereas the single guide RNA （sgRNA） is typically expressed from a Pol III promoter, such as U6 or U3 promoter.展开更多
The creation of new soybean varieties has been limited by genomic duplication and redundancy.Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic ...The creation of new soybean varieties has been limited by genomic duplication and redundancy.Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)systems are promising strategies for overcoming these obstacles.CRISPR/Cpf1 is a robust tool for multiplex gene editing.However,large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species.Here,we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting.The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA(crRNA)array.The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter.The optimized system exhibited editing efficiencies of up to 91.7%.Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed,with an efficiency of up to 17.1%.We successfully employed CRISPR/LbCpf1 to produce small fragments(<1 Kb)and large chromosomal segment deletions(10 Kb-1 Mb)involving four different gene clusters in soybean.Together,these data demonstrate the power of the CRISPR/LbCpf1 platform for multiplex gene editing and chromosomal segment deletion in soybean,supporting the use of this technology in both basic research and agricultural applications.展开更多
Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease...Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.展开更多
Prime editing(PE)technology enables precise alterations in the genetic code of a genome of interest.PE offers great potential for identifying major agronomically important genes in plants and editing them into superio...Prime editing(PE)technology enables precise alterations in the genetic code of a genome of interest.PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants,ideally targeting multiple loci simultaneously to realize the collective effects of the edits.Here,we report the development of a modular assembly-based multiplex PE system in rice and demon-strate its efficacy in editing up to four genes in a single transformation experiment.The duplex PE(DPE)system achieved a co-editing efficiency of 46.1%in the T0 generation,converting TFIIAg5 to xa5 and xa23 to Xa23SW11.The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae(Xoo)strains in the T1 generation.In addition,we success-fully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele,achieving a co-editing rate of 57.14%.Furthermore,with the quadruple PE(QPE)system,we edited four genes—two for herbicide tolerance(OsEPSPS1 and OsALS1)and two for Xoo resistance(TFIIAg5 and OsSWEET11a)—using one construct,with a co-editing efficiency of 43.5%for all four genes in the T0 gen-eration.We performed multiplex PE usingfive more constructs,including two for triplex PE(TPE)and three for QPE,each targeting a different set of genes.The editing rates were dependent on the activity of pegRNA and/or ngRNA.For instance,optimization of ngRNA increased the PE rates for one of the targets(OsSPL13)from 0%to 30%but did not improve editing at another target(OsGS2).Overall,our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents,making it feasible for more labs to utilize PE for their editing experiments.Thesefindings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.展开更多
Aquaporins play important regulatory roles in improving plant abiotic stress tolerance.To better understand whether the Os PIP1 genes collectively dominate the osmotic regulation in rice under salt stress,a cluster ed...Aquaporins play important regulatory roles in improving plant abiotic stress tolerance.To better understand whether the Os PIP1 genes collectively dominate the osmotic regulation in rice under salt stress,a cluster editing of the Os PIP1;1,Os PIP1;2 and Os PIP1;3 genes in rice was performed by CRISPR/Cas9 system.Sequencing showed that two mutants with Cas9-free,line 14 and line 18 were successfully edited.Briefly,line 14 deleted a single C base in both the Os PIP1;1 and Os PIP1;3 genes,and inserted a single T base in the Os PIP1;2 gene,respectively.While line 18 demonstrated an insertion of a single A base in the Os PIP1;1gene and a single T base in both the Os PIP1;2 and Os PIP1;3 genes,respectively.Multiplex editing of the Os PIP1 genes significantly inhibited photosynthetic rate and accumulation of compatible metabolites,but increased MDA contents and osmotic potentials in the mutants,thus delaying rice growth under salt stress.Functional loss of the Os PIP1 genes obviously suppressed the expressions of the Os PIP1,Os SOS1,Os CIPK24 and Os CBL4 genes,and increased the influxes of Na+and effluxes of K^(+)/H^(+)in the roots,thus accumulating more Na+in rice mutants under salt stress.This study suggests that the Os PIP1 genes are essential modulators collectively contributing to the enhancement of rice salt stress tolerance,and multiplex editing of the Os PIP1 genes provides insight into the osmotic regulation of the PIP genes.展开更多
Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems.Inevitably,researchers need to pursue the co-modification...Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems.Inevitably,researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates.Base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1.Since multiplexed application of base editing is still limited by its low throughput procedure,we thus here develop a rapid and efficient multiplex base editing system in S.oneidensis.Four approaches to express multiple gRNAs were assessed firstly,and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster.Then,a smart scheme was designed to deliver one-pot assembly of multiple gRNAs.3,5,and 8 genes were deactivated using this system with editing efficiency of 83.3%,100%and 12.5%,respectively.To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette,different promoters,handles,and terminators were screened.This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain.The maximum power density of the multiplex engineered strain HRF(8BE)in microbial fuel cells was 1108.1 mW/m2,which was 21.67 times higher than that of the wild-type strain.This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella,and may provide valuable insights in fundamental and applied research of extracellular electron transfer.展开更多
Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.A...Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.Although CRISPR/Cas9 technology has been applied in Y.lipolytica,achieving a consistently high editing performance re-mains challenging owing to the low homologous recombination efficiency and variability in system components.In this study,we optimized CRISPR/Cas9-mediated genome editing in Y.lipolytica to enhance its editing effi-ciency.Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression,we achieved a gene disruption efficiency of 92.5%.The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5%.KU70 deletion in the Cas9 system increased the integration efficiency to 92.5%,and Rad52 and Sae2 overexpression boosted homologous recombination.The introduction of Cas9D147Y,P411T(iCas9)enhanced the efficiency of both gene disruption and genome integration.This study provides a powerful tool for efficient gene editing in Y.lipolytica,which will accelerate the construction of yeast cell factories.展开更多
The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/C...The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap- tion of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/ Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac- tors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.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
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various organisms.With efficient...The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various organisms.With efficient and proper expression of multiple guide RNAs(gRNAs),the CRISPR/Cas system is particularly suitable for multiplex genome editing.During the past several years,different CRISPR/Cas expression strategies,such as two-component transcriptional unit,single transcriptional unit,and bidirectional promoter systems,have been developed to efficiently express gRNAs as well as Cas nucleases.Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes,endogenous RNases,and exogenous endoribonuclease(Csy4).Besides being constitutively and ubiquitously expressed,inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible,tissue-specific,and/or synthetic promoters for specific research purposes.Most recently,the emergence of CRISPR/Cas ribonucleoprotein delivery methods,such as engineered nanoparticles,further revolutionized transgene-free and multiplex genome editing.In this review,we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.展开更多
Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system,also known as CRISPR/Cpf1,has been successfully harnessed for genome engineering in many plants,but not in grapevine yet.Here we develope...Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system,also known as CRISPR/Cpf1,has been successfully harnessed for genome engineering in many plants,but not in grapevine yet.Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006(LbCas12a)in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1(TMT1)and dihydroflavonol-4-reductase 1(DFR1)genes in 41B cells.Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells.Heat treatment(34℃)improved the editing efficiencies of CRISPR/LbCas12a system,and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3%to 44.6%and 29.9%to 37.3%after heat treatment,respectively.Moreover,the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing.In addition,genome editing with truncated crRNAs(trucrRNAs)showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis,whereas trucrRNAs with shorter regions of target complementarity≤18 nt in length may not induce detectable mutations in 41B cells.All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.展开更多
基金supported by the Biological BreedingMajor Projects(2023ZD04076)the National Natural Science Foundation of China(32300312)+2 种基金the Innovation Program of Chinses Academy of Agricultural Sciences(CAAS-CSIAF-202303)the Guangdong Basic and Applied Basic Research Foundation(2020B1515120086)the KeyArea Research and Development Program of Guangdong Province(2021B0707010006)。
文摘Rice, a global staple food, is critical for food security. The cultivated Oryza sativa, domesticated from wild O. rufipogon, derives~80%of its 993 identified domestication-related genes from O. rufipogon and 20%from South/Southeast Asian wild O. nivara(Jing et al., 2023). Genes like An-1, BH4, PROG1,SH4, Rc, Rd, and GS3—which regulate awn length, hull color,til er angle, seed shattering, pericarp color, seed length, and thousand-grain weight, respectively—were selected against during domestication to form modern O. sativa(Yu et al., 2021).However, domestication and yield-focused breeding eliminated wild rice's valuable genes(e.g., for disease resistance, stress tolerance, nutrition), narrowing genetic diversity and impeding efforts to meet growing societal demands.
基金supported by the Agricultural Science and Technology Innovation Program
文摘Generating mutants bearing multiple gene modifications is essential for determining the functions of gene family members with redundant functions, or for analyzing epistatic re- lationships in genetic pathways. Using conventional methods, mutants with multiple gene mutations are generated by several rounds of intercrossing plants carrying a single mutation and identification of the offspring. This process is both timeconsuming and labor-intensive. Moreover, if the genes of interest are closely linked, multiple mutations can not be generated （Wijnker and de Jong, 2008）.
基金supported by Genetically Modified Breeding Major Projects(No.2016ZX08010-002-008)the National Natural Science Foundation of China(Nos.31501239 and 31401454)
文摘Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.
基金supported by the National Natural Science Foundation of China(32001532 and 31860411)the National Key Research and Development Program of China,(2022YFF1000020)+1 种基金Hunan Seed Industry Innovation Project(2021NK1012)the Yunnan Tobacco Company Project(2020530000241009)。
文摘The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.
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supported by grants from the Biological Breeding-National Science and Technology Major Project(2022ZD04011,2023ZD04073)National Natural Science Foundation of China(32325035,32071864)+4 种基金Fundamental Research Funds for Central Non-profit Scientific Institution(1610392024008)Central Laboratory of Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Hainan Seed Industry Laboratory(B23CJ0208)Yunnan Academy of Tobacco Agricultural Sciences(CNTC-YC2022530000241013,CNTC110201501015(JY-02))Hohhot Key R&D Project(2023-JBGS-S-1)Inner Mongolia Center of Pratacultural Technology Innovation(CCPTZX2023B01).
文摘CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(Medicago sativa L.),the most important forage legume worldwide,remains a formidable challenge.Here,we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves.We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system.Subsequently,we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA(PTG)expression cassettes,each consisting of three tRNA-sgRNA units,to simultaneously edit three or four alfalfa genes,coupled with the visual reporter RH1 or RUBY.This toolkit showed efficient multiplex editing in the hairy root system with visual selection.We successfully obtained regenerated,red-colored shoots resulting from the stable transformation of alfalfa.These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa.Our improved CRISPR/Cas9 multiplex system enables convenient,high-efficiency multiplex genome editing in alfalfa,providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.
基金supported by the National Natural Science Foundation of China(grant numbers 31971617 and 32022055)the Science and Technology Innovation 2030-Major Project(grant number 2023ZD04057)the Zhejiang A&F University Starting Funding(2021FR026).
文摘Dear Editor,Receptor-like kinases(RLKs)constitute a large superfamily that regulates diverse biological processes in plants(De Smet et al.,2009).In the perennial tree Populus,nearly 200 RLKs are highly expressed in developing xylem,suggesting important roles in wood formation(Song et al.,2011;Zan et al.,2013;Xie et al.,2023).
基金supported by the National Drug Innovation Major Project (2018ZX09711001-006-012)the National Natural Science Foundation of China (31770088,31570072 and 31430004)+2 种基金the Science and Technology Commission of Shanghai Municipality(18ZR1446700)the Derivative Bank of Chinese Biological Resources,CAS(ZSYS-016)Shanghai Engineering Research Center of Plant Germplasm Resources (17DZ2252700)。
文摘CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination(HR) ability. Here, a deaminase-assisted base editor d Cas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9(dCas9), the cytidine deaminase from Petromyzon marinus(PmCDA1), the uracil DNA glycosylase inhibitor(UGI) and the protein degradation tag(LVA tag). Using d Cas9-CDA-ULstr , we achieved single-, double-and triple-point mutations(cytosine-to-thymine substitutions)at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes(with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the d Cas9-CDA-ULstr -based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.
文摘The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl （also known as Cas12a） system is a two-component transcriptional unit （TCTU） in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase （pol） II promoter, whereas the single guide RNA （sgRNA） is typically expressed from a Pol III promoter, such as U6 or U3 promoter.
基金supported by grants from National Science Foundation of China(NSFC31901957)Fundamental Research Funds for the Central Universities(JCQY201903)。
文摘The creation of new soybean varieties has been limited by genomic duplication and redundancy.Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)systems are promising strategies for overcoming these obstacles.CRISPR/Cpf1 is a robust tool for multiplex gene editing.However,large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species.Here,we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting.The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA(crRNA)array.The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter.The optimized system exhibited editing efficiencies of up to 91.7%.Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed,with an efficiency of up to 17.1%.We successfully employed CRISPR/LbCpf1 to produce small fragments(<1 Kb)and large chromosomal segment deletions(10 Kb-1 Mb)involving four different gene clusters in soybean.Together,these data demonstrate the power of the CRISPR/LbCpf1 platform for multiplex gene editing and chromosomal segment deletion in soybean,supporting the use of this technology in both basic research and agricultural applications.
基金grants from the National Natural Science Foundation of China (91435203 and 31991222)the Major Program of Guangdong Basic and Applied Basic Research (2019B030302006)。
文摘Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.
基金supported by an NSF award (IOS-2210259 to B.Y.)a subaward to the University of Missouri from the Heinrich Heine University of Dusseldorf funded by the Bill&Melinda Gates Foundation (OPP1155704)supported by the Daniel Millikan Award for Outstanding Research in Plant–Microbe Interactions at the University of Missouri.
文摘Prime editing(PE)technology enables precise alterations in the genetic code of a genome of interest.PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants,ideally targeting multiple loci simultaneously to realize the collective effects of the edits.Here,we report the development of a modular assembly-based multiplex PE system in rice and demon-strate its efficacy in editing up to four genes in a single transformation experiment.The duplex PE(DPE)system achieved a co-editing efficiency of 46.1%in the T0 generation,converting TFIIAg5 to xa5 and xa23 to Xa23SW11.The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae(Xoo)strains in the T1 generation.In addition,we success-fully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele,achieving a co-editing rate of 57.14%.Furthermore,with the quadruple PE(QPE)system,we edited four genes—two for herbicide tolerance(OsEPSPS1 and OsALS1)and two for Xoo resistance(TFIIAg5 and OsSWEET11a)—using one construct,with a co-editing efficiency of 43.5%for all four genes in the T0 gen-eration.We performed multiplex PE usingfive more constructs,including two for triplex PE(TPE)and three for QPE,each targeting a different set of genes.The editing rates were dependent on the activity of pegRNA and/or ngRNA.For instance,optimization of ngRNA increased the PE rates for one of the targets(OsSPL13)from 0%to 30%but did not improve editing at another target(OsGS2).Overall,our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents,making it feasible for more labs to utilize PE for their editing experiments.Thesefindings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.
基金supported by the National Key Research and Development Program of China (2021YFF1000402-2)the Key Project of Transgenic Crops Cultivation (2016ZX08010005-9)。
文摘Aquaporins play important regulatory roles in improving plant abiotic stress tolerance.To better understand whether the Os PIP1 genes collectively dominate the osmotic regulation in rice under salt stress,a cluster editing of the Os PIP1;1,Os PIP1;2 and Os PIP1;3 genes in rice was performed by CRISPR/Cas9 system.Sequencing showed that two mutants with Cas9-free,line 14 and line 18 were successfully edited.Briefly,line 14 deleted a single C base in both the Os PIP1;1 and Os PIP1;3 genes,and inserted a single T base in the Os PIP1;2 gene,respectively.While line 18 demonstrated an insertion of a single A base in the Os PIP1;1gene and a single T base in both the Os PIP1;2 and Os PIP1;3 genes,respectively.Multiplex editing of the Os PIP1 genes significantly inhibited photosynthetic rate and accumulation of compatible metabolites,but increased MDA contents and osmotic potentials in the mutants,thus delaying rice growth under salt stress.Functional loss of the Os PIP1 genes obviously suppressed the expressions of the Os PIP1,Os SOS1,Os CIPK24 and Os CBL4 genes,and increased the influxes of Na+and effluxes of K^(+)/H^(+)in the roots,thus accumulating more Na+in rice mutants under salt stress.This study suggests that the Os PIP1 genes are essential modulators collectively contributing to the enhancement of rice salt stress tolerance,and multiplex editing of the Os PIP1 genes provides insight into the osmotic regulation of the PIP genes.
基金supported by the National Key Research and Development Program of China (2018YFA0901300)the National Natural Science Foundation of China (NSFC 32071411,NSFC 22078240,and NSFC 21621004)the Young Elite Scientists Sponsorship Program by Tianjin (TJSQNTJ-2018-16).
文摘Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems.Inevitably,researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates.Base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1.Since multiplexed application of base editing is still limited by its low throughput procedure,we thus here develop a rapid and efficient multiplex base editing system in S.oneidensis.Four approaches to express multiple gRNAs were assessed firstly,and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster.Then,a smart scheme was designed to deliver one-pot assembly of multiple gRNAs.3,5,and 8 genes were deactivated using this system with editing efficiency of 83.3%,100%and 12.5%,respectively.To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette,different promoters,handles,and terminators were screened.This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain.The maximum power density of the multiplex engineered strain HRF(8BE)in microbial fuel cells was 1108.1 mW/m2,which was 21.67 times higher than that of the wild-type strain.This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella,and may provide valuable insights in fundamental and applied research of extracellular electron transfer.
基金supported by the National Natural Science Founda-tion of China(U23A20268)the National Natural Science Foundation of Shandong Province(ZR2022ZD24)the Taishan Scholar Project of Shandong Province(tsqn202312061).
文摘Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility.Efficient genome editing tools are essential for advancing its biotechnological applications.Although CRISPR/Cas9 technology has been applied in Y.lipolytica,achieving a consistently high editing performance re-mains challenging owing to the low homologous recombination efficiency and variability in system components.In this study,we optimized CRISPR/Cas9-mediated genome editing in Y.lipolytica to enhance its editing effi-ciency.Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression,we achieved a gene disruption efficiency of 92.5%.The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5%.KU70 deletion in the Cas9 system increased the integration efficiency to 92.5%,and Rad52 and Sae2 overexpression boosted homologous recombination.The introduction of Cas9D147Y,P411T(iCas9)enhanced the efficiency of both gene disruption and genome integration.This study provides a powerful tool for efficient gene editing in Y.lipolytica,which will accelerate the construction of yeast cell factories.
文摘The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap- tion of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/ Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac- tors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.
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This work was supported by NSF Plant Genome Research Project Grant(1740874)the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project PEN04659 and Accession#1016432.
文摘The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various organisms.With efficient and proper expression of multiple guide RNAs(gRNAs),the CRISPR/Cas system is particularly suitable for multiplex genome editing.During the past several years,different CRISPR/Cas expression strategies,such as two-component transcriptional unit,single transcriptional unit,and bidirectional promoter systems,have been developed to efficiently express gRNAs as well as Cas nucleases.Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes,endogenous RNases,and exogenous endoribonuclease(Csy4).Besides being constitutively and ubiquitously expressed,inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible,tissue-specific,and/or synthetic promoters for specific research purposes.Most recently,the emergence of CRISPR/Cas ribonucleoprotein delivery methods,such as engineered nanoparticles,further revolutionized transgene-free and multiplex genome editing.In this review,we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.
基金Open access funding provided by Shanghai Jiao Tong Universitysupported by grants from the Strategic Priority Research Program of Chinese Academy of Sciences(XDA24030404-3)+2 种基金the National Natural Science Foundation of China(32001994)the Agricultural Breeding Project of Ningxia Hui Autonomous Region(NXNYYZ20210104)the Youth Innovation Promotion Association CAS(2022078).
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system,also known as CRISPR/Cpf1,has been successfully harnessed for genome engineering in many plants,but not in grapevine yet.Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006(LbCas12a)in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1(TMT1)and dihydroflavonol-4-reductase 1(DFR1)genes in 41B cells.Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells.Heat treatment(34℃)improved the editing efficiencies of CRISPR/LbCas12a system,and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3%to 44.6%and 29.9%to 37.3%after heat treatment,respectively.Moreover,the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing.In addition,genome editing with truncated crRNAs(trucrRNAs)showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis,whereas trucrRNAs with shorter regions of target complementarity≤18 nt in length may not induce detectable mutations in 41B cells.All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.
