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
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
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
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.
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.
