CRISPR/Cas （clustered regularly interspaced short palindromic repeats/CR1SPR-associated proteins） is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently,...CRISPR/Cas （clustered regularly interspaced short palindromic repeats/CR1SPR-associated proteins） is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic To plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67 %. Stable knockout transformants for maize phytoene synthase gene （PSYI） were obtained. Mutations occurred in germ ceils can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.展开更多
High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 syst...High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.展开更多
Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adj...Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adjacent motif （PAM） at the 5＇ end of target sequences. In addition, Cpfl requires only a single shorter crRNA and cleaves DNA in a staggered fashion with 5＇ overhangs （Zetsche et al., 2015）.展开更多
CRISPR （clustered regularly interspaced short palindromic repeats）-Cas9-based genome editing has revolutionized func- tional genomics in many biological research fields. The specificity and potency of CR1SPR-Cas9 ge...CRISPR （clustered regularly interspaced short palindromic repeats）-Cas9-based genome editing has revolutionized func- tional genomics in many biological research fields. The specificity and potency of CR1SPR-Cas9 genome editing make it ideal for investigating the function of genes in vivo （Hsu et al., 2014）. Gene duplication is a key driver of evolu- tionary novelty （Taylor and Raes, 2004）. However, duplicated genes with near-identical sequences and functional redun- dancy have posed challenges for genetic analysis （Woollard, 2005）. The functions of duplicated genes can be assessed by simultaneous knockdown using homology-based methods such as RNA interference （RNAi） （Tischler et al., 2006）, Generation of double or triple mutants is an alternative way to assess functional redundancy of duplicated genes, However, generation of such compound mutants by forward or reverse genetic methods is time consuming.展开更多
Dear Editor,Rice(Oryza sativa L.),one of the most important staple crops,feeds more than half of the world’s population.The appli-cation of nitrogen(N)fertilizer has greatly enhanced rice production but has also incr...Dear Editor,Rice(Oryza sativa L.),one of the most important staple crops,feeds more than half of the world’s population.The appli-cation of nitrogen(N)fertilizer has greatly enhanced rice production but has also increased the cost of rice production and environmental pollution.Based on previous research,O.sativa subsp.japonica rice varieties generally have lower nitro-gen use efficiency(NUE)than O.sativa subsp.indica rice varieties(Zhang and Chu 2020).Therefore,breeding new japonica rice varieties that not only produce high yields,have superior taste and texture,and possess disease resistance but also exhibit improved NUE is increasingly essential.展开更多
Genome editing has the potential to enhance yield and quality traits of crops.However,standard genetic transformation methods are not always applicable to modern germplasm.To tackle this challenge in the widely cultiv...Genome editing has the potential to enhance yield and quality traits of crops.However,standard genetic transformation methods are not always applicable to modern germplasm.To tackle this challenge in the widely cultivated variety Ligena of the oilseed crop camelina(Camelina sativa(L.)Crantz),an only recently established principle of adventitious shoot formation from immature zygotic embryos was employed to further improve its fatty acid profile.In this approach,the three subgenomic homeologs of the FATTY ACID ELONGASE 1(FAE1)gene were subjected to targeted mutagenesis.To pre-validate the Cas9-interacting,target motif-specific guide(g)RNAs,a robust protoplast-based DNA transfection method was established.This assay demonstrated that the preselected gRNAs were capable of eliciting mutations across all three camelina FAE1 homeologs.Likewise,targeted mutagenesis was successful at the whole-plant level.Triple-homozygous fae1 knockout mutants were identified amongst a segregating generation M_(3) family.Gas chromatography of lipid extracts from M4 seeds revealed a significant increase in all unsaturated C18 fatty acids including the particularly valuable a-linolenic acid.This was accompanied by a near elimination of the C20 and C22 very long-chain fatty acids including the nutritionally problematic erucic acid.Altogether,we have developed camelina elite lines with two significantly improved properties of high relevance for a health-promoting human nutrition.展开更多
Mutations,serving as the raw materials of evolution,have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions.Global and targeted mutagenesis are two main m...Mutations,serving as the raw materials of evolution,have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions.Global and targeted mutagenesis are two main methods of obtaining various mutations,distinguished by the range of action they can cover.While the former one stresses the mining of novel genetic loci within the whole genomic background,targeted mutagenesis performs in a more straightforward manner,bringing evolutionary escape and error catastrophe under control.In this review,we classify the existing techniques of targeted mutagenesis into two categories in terms of whether the diversity is generated in vitro or in vivo,and briefly introduce the mechanisms and applications of them separately.The inherent connections and development trends of the two classes are also discussed to provide an insight into the next generation evolution research.展开更多
Lactococcus lactis serves as an important platform for heterologous protein production,with the nisin-controlled gene expression(NICE)system being widely employed for regulated protein overexpression.However,the NICE ...Lactococcus lactis serves as an important platform for heterologous protein production,with the nisin-controlled gene expression(NICE)system being widely employed for regulated protein overexpression.However,the NICE system relies on the native RNA polymerase,which limits transcriptional efficiency,and there remains a lack of tools enabling continuous target gene mutagenesis in L.lactis.In this study,we enhanced the NICE system by integrating the highly processive T7 RNA polymerase(T7RNAP)to boost protein expression.A theophylline-dependent riboswitch,RbxE,was incorporated into the nisin-induced promoter to mitigate the toxicity caused by basal T7RNAP expression in Escherichia coli.Directed mutagenesis of the riboswitch region between the stem-loop and the ribosome binding site optimized T7RNAP expression,leading to a 2.4-fold increase upon nisin and theophylline induction in L.lactis.The resulting NICE-T7 system achieved a 2.8-fold increase in GFP compared to the original NICE system.Furthermore,adenosine deaminase TadA8e was fused to T7RNAP to generate the MutaT7LL system,facilitating targeted A-to-G mutagenesis and successfully reactivated an erythromycin resis-tance gene with a mutation efficiency of 1.33×10^(-6).Overall,this study presents an upgraded NICE system that enhances protein production and enables continuous in vivo mutagenesis of target genes in L.lactis.展开更多
The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation ...The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation efficiency induced by the CRISPR/Cas9 system in many plant species.However,a heat stress treatment has not been applied during the tissue culture process in citrus.To develop an efficient heat stress regime to improve the efficiency of CRISPR/Cas9-mediated targeted mutagenesis,three and five cycles of heat stress treatments were used during callus induction in citrus.The results showed that the heat stress treatment with three cycles of 24 h at 37℃,followed by 24 h at 26℃,increased the mutation efficiency by 11.6%compared with no heat stress treatment,and that five cycles of heat stress treatment were optimal,from which 50%mutants had a 100%mutation rate.The mutation profiles of Cas9 at 28℃ for 10 d and 37℃ for three or five cycles were similar,indicating that heat stress treatment did not affect the non-homologous end joining repair pathway.No detectable off-target mutation was detected in the potential off-target sites with four nucleotide mismatches compared with the designed on-target site.This study demonstrated that five cycles of heat stress treatment during callus induction could efficiently increase the mutation efficiency mediated by the CRISPR/Cas9 system without observable negative effects,and provided an efficient Cas9-mediated citrus genome editing system to produce mutants with a high mutation rate.展开更多
Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively e...Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.展开更多
The recent discovery of the mode of action of the CRISPR/Cas9 systemhas provided biologists with a useful tool for generating site-specific mutations in genes of interest.In plants,site-targeted mutations are usually ...The recent discovery of the mode of action of the CRISPR/Cas9 systemhas provided biologists with a useful tool for generating site-specific mutations in genes of interest.In plants,site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome.The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs,including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA,and the sequence of the Cas9 nuclease itself.To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana,we investigated several features of its nucleotide and/or amino acid sequence,including the codon usage,the number of nuclear localization signals(NLSs),and the presence or absence of introns.We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one.However,the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence,which dramatically improved the editing efficiency of the constructs.None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype,whereas between 70%and 100%of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes.The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.展开更多
基金supported by the grants from the National Natural Science Foundation of China(Nos.31225020,31421005,and 91435206)grant from the National High Technology Research and Development of China(No.2012AA10A305)2011-G15 from the‘948’project
文摘CRISPR/Cas （clustered regularly interspaced short palindromic repeats/CR1SPR-associated proteins） is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic To plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67 %. Stable knockout transformants for maize phytoene synthase gene （PSYI） were obtained. Mutations occurred in germ ceils can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.
基金financially supported by National Key Research and Development Program of China(2016YFD0100501)the National Natural Science Foundation of China(31871241,31371233)+3 种基金the Natural Science Foundation of Jiangsu Province(BE2017345,PZCZ201702,BE2018351)the Research and Innovation Program of Postgraduate in Jiangsu Province(KYCX17_1886)the Priority Academic Program Development of Jiangsu Higher Education Institutionsthe Yangzhou University International Academic Exchange Fund。
文摘High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.
基金supported by the National Natural Science Foundation of China(No.3140101312)the Zhejiang Provincial Natural Science Foundation of China(No.LZ14C130003)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences
文摘Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adjacent motif （PAM） at the 5＇ end of target sequences. In addition, Cpfl requires only a single shorter crRNA and cleaves DNA in a staggered fashion with 5＇ overhangs （Zetsche et al., 2015）.
基金supported by National Institutes of Health(NIH grant R01GM054657)to A.D.C
文摘CRISPR （clustered regularly interspaced short palindromic repeats）-Cas9-based genome editing has revolutionized func- tional genomics in many biological research fields. The specificity and potency of CR1SPR-Cas9 genome editing make it ideal for investigating the function of genes in vivo （Hsu et al., 2014）. Gene duplication is a key driver of evolu- tionary novelty （Taylor and Raes, 2004）. However, duplicated genes with near-identical sequences and functional redun- dancy have posed challenges for genetic analysis （Woollard, 2005）. The functions of duplicated genes can be assessed by simultaneous knockdown using homology-based methods such as RNA interference （RNAi） （Tischler et al., 2006）, Generation of double or triple mutants is an alternative way to assess functional redundancy of duplicated genes, However, generation of such compound mutants by forward or reverse genetic methods is time consuming.
基金supported by the STI 2030-Major Projects(2023ZD04066)the Jiangsu Seed Industry Revitalization Project(JBGS[2021]011 and JBGS[2021]039)+1 种基金the Ministry of Agriculture and Rural Affairs of China,the Jiangsu Provincial Key Research and Development Program(BE2021374 and BE2020339)the National Natural Science Foundation of China(32201898).
文摘Dear Editor,Rice(Oryza sativa L.),one of the most important staple crops,feeds more than half of the world’s population.The appli-cation of nitrogen(N)fertilizer has greatly enhanced rice production but has also increased the cost of rice production and environmental pollution.Based on previous research,O.sativa subsp.japonica rice varieties generally have lower nitro-gen use efficiency(NUE)than O.sativa subsp.indica rice varieties(Zhang and Chu 2020).Therefore,breeding new japonica rice varieties that not only produce high yields,have superior taste and texture,and possess disease resistance but also exhibit improved NUE is increasingly essential.
基金funded by the German Federal Ministry of Education and Research(BMBF),grant FKZ031B0343A and Cthe German Research Foundation(Deutsche Forschungsgemeinschaft)grant 491250510.
文摘Genome editing has the potential to enhance yield and quality traits of crops.However,standard genetic transformation methods are not always applicable to modern germplasm.To tackle this challenge in the widely cultivated variety Ligena of the oilseed crop camelina(Camelina sativa(L.)Crantz),an only recently established principle of adventitious shoot formation from immature zygotic embryos was employed to further improve its fatty acid profile.In this approach,the three subgenomic homeologs of the FATTY ACID ELONGASE 1(FAE1)gene were subjected to targeted mutagenesis.To pre-validate the Cas9-interacting,target motif-specific guide(g)RNAs,a robust protoplast-based DNA transfection method was established.This assay demonstrated that the preselected gRNAs were capable of eliciting mutations across all three camelina FAE1 homeologs.Likewise,targeted mutagenesis was successful at the whole-plant level.Triple-homozygous fae1 knockout mutants were identified amongst a segregating generation M_(3) family.Gas chromatography of lipid extracts from M4 seeds revealed a significant increase in all unsaturated C18 fatty acids including the particularly valuable a-linolenic acid.This was accompanied by a near elimination of the C20 and C22 very long-chain fatty acids including the nutritionally problematic erucic acid.Altogether,we have developed camelina elite lines with two significantly improved properties of high relevance for a health-promoting human nutrition.
基金This work was supported by the National Natural Science Foundation of China(NSFC 21627812&21676156).
文摘Mutations,serving as the raw materials of evolution,have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions.Global and targeted mutagenesis are two main methods of obtaining various mutations,distinguished by the range of action they can cover.While the former one stresses the mining of novel genetic loci within the whole genomic background,targeted mutagenesis performs in a more straightforward manner,bringing evolutionary escape and error catastrophe under control.In this review,we classify the existing techniques of targeted mutagenesis into two categories in terms of whether the diversity is generated in vitro or in vivo,and briefly introduce the mechanisms and applications of them separately.The inherent connections and development trends of the two classes are also discussed to provide an insight into the next generation evolution research.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110200)the National Natural Science Foundation of China(32270031)+3 种基金the National Science Foundation for Distinguished Young Scholars(32225031)NSFC Basic Science Center Program(32488301)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-CXRC-064)the Hundred Talents Program of the Chinese Academy of Sciences.
文摘Lactococcus lactis serves as an important platform for heterologous protein production,with the nisin-controlled gene expression(NICE)system being widely employed for regulated protein overexpression.However,the NICE system relies on the native RNA polymerase,which limits transcriptional efficiency,and there remains a lack of tools enabling continuous target gene mutagenesis in L.lactis.In this study,we enhanced the NICE system by integrating the highly processive T7 RNA polymerase(T7RNAP)to boost protein expression.A theophylline-dependent riboswitch,RbxE,was incorporated into the nisin-induced promoter to mitigate the toxicity caused by basal T7RNAP expression in Escherichia coli.Directed mutagenesis of the riboswitch region between the stem-loop and the ribosome binding site optimized T7RNAP expression,leading to a 2.4-fold increase upon nisin and theophylline induction in L.lactis.The resulting NICE-T7 system achieved a 2.8-fold increase in GFP compared to the original NICE system.Furthermore,adenosine deaminase TadA8e was fused to T7RNAP to generate the MutaT7LL system,facilitating targeted A-to-G mutagenesis and successfully reactivated an erythromycin resis-tance gene with a mutation efficiency of 1.33×10^(-6).Overall,this study presents an upgraded NICE system that enhances protein production and enables continuous in vivo mutagenesis of target genes in L.lactis.
基金supported by the National Key Research and Development Program of China(Grant No.2022YFD1201600)Earmarked Fund for China Agriculture Research System(Grant No.CARS-26)+1 种基金Chongqing Natural Science Foundation Project(Grant No.CSTB2023NSCQ-MSX1085)Cultivar Improvement of Nanfeng Orange.
文摘The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome.The efficiency of Cas9-mediated genome editing is temperature sensitive.A high-temperature regime can increase the mutation efficiency induced by the CRISPR/Cas9 system in many plant species.However,a heat stress treatment has not been applied during the tissue culture process in citrus.To develop an efficient heat stress regime to improve the efficiency of CRISPR/Cas9-mediated targeted mutagenesis,three and five cycles of heat stress treatments were used during callus induction in citrus.The results showed that the heat stress treatment with three cycles of 24 h at 37℃,followed by 24 h at 26℃,increased the mutation efficiency by 11.6%compared with no heat stress treatment,and that five cycles of heat stress treatment were optimal,from which 50%mutants had a 100%mutation rate.The mutation profiles of Cas9 at 28℃ for 10 d and 37℃ for three or five cycles were similar,indicating that heat stress treatment did not affect the non-homologous end joining repair pathway.No detectable off-target mutation was detected in the potential off-target sites with four nucleotide mismatches compared with the designed on-target site.This study demonstrated that five cycles of heat stress treatment during callus induction could efficiently increase the mutation efficiency mediated by the CRISPR/Cas9 system without observable negative effects,and provided an efficient Cas9-mediated citrus genome editing system to produce mutants with a high mutation rate.
基金supported by the UK Medical Research Council and the European Research Council (DARCGENs, No. 249869)
文摘Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.
基金supported by core funding of the IPBsupported by grant STU642-1/1(DFG/GRC,Deutsche Forschungsgemeinschaft)supported by the National Science Foundation MCB award 1516371 to C.L.-P.and E.C.
文摘The recent discovery of the mode of action of the CRISPR/Cas9 systemhas provided biologists with a useful tool for generating site-specific mutations in genes of interest.In plants,site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome.The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs,including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA,and the sequence of the Cas9 nuclease itself.To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana,we investigated several features of its nucleotide and/or amino acid sequence,including the codon usage,the number of nuclear localization signals(NLSs),and the presence or absence of introns.We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one.However,the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence,which dramatically improved the editing efficiency of the constructs.None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype,whereas between 70%and 100%of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes.The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.
