MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both acade...MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.展开更多
CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable...CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.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
Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of ...Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.展开更多
The seeds for genomics were sown with the development of DNA sequencing(Sanger et al., 1977), cultivated with each advance in molecular biology, and have since grown into one of the most important aspects of the life ...The seeds for genomics were sown with the development of DNA sequencing(Sanger et al., 1977), cultivated with each advance in molecular biology, and have since grown into one of the most important aspects of the life sciences. With the sequencing of the first draft human genome(Venter et al.,2001), the era of post-genomics began. Once one genome has been sequenced, it can be used as the reference for a certain species, and sequences from individuals can be mapped onto it to compare genetic variations among different lines.展开更多
The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops.Here,we established efficient Agrobacterium-mediated clustered regularly...The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops.Here,we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)genome editing in a perennial,stress-tolerant forage grass,sheepgrass(Leymus chinensis).By screening for active single-guide RNAs(sg RNAs),accessions that regenerate well,suitable Agrobacterium strains,and optimal culture media,and co-expressing the morphogenic factor Ta WOX5,we achieved 11%transformation and 5.83%editing efficiency in sheepgrass.Knocking out Teosinte Branched1(TB1)significantly increased tiller number and biomass.This study opens avenues for studying gene function and breeding in sheepgrass.展开更多
Dear Editor,The CRISPR-Cas9（clustered regularly interspaced short palindromic repeats/Cas9）system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement a...Dear Editor,The CRISPR-Cas9（clustered regularly interspaced short palindromic repeats/Cas9）system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs（PAMs）.To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs and v variants with altered PAM specificities （Kleinstiver et al., 2015; Hu et al., 2016）.展开更多
Ongoing advances in genomics and genetics have led researchers to highlight the significant role of large DNA structural variants(SVs)in genetic diversity.SVs are genomic sequence variants of various types,including i...Ongoing advances in genomics and genetics have led researchers to highlight the significant role of large DNA structural variants(SVs)in genetic diversity.SVs are genomic sequence variants of various types,including insertions,deletions,inversions,translocations,and copy number variants(CNVs or duplications).These SVs,which are usually of 1 kb or more,can have profound impacts at both the individual and species level.They are also often linked to the initiation and progression of complex genetic diseases(Stankiewicz and Lupski,2010).展开更多
Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regu...Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.展开更多
Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the ac...Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.展开更多
Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transitio...Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.展开更多
Virus infection poses a constant threat on global crop productivity.It is estimated that virus diseases account for at least 10% of crop losses worldwide.Development of resistant crops using naturally evolved resistan...Virus infection poses a constant threat on global crop productivity.It is estimated that virus diseases account for at least 10% of crop losses worldwide.Development of resistant crops using naturally evolved resistance genes has been a main measure for controlling plant virus epidemics.However,isolation of plant resistance genes is costly and time-consuming.Furthermore,most plant resistance genes are pathogen race-specific,with very few of them being broadly specific (Langner et al.,2018).Consequently,artificial immunity with engineered resistance to virus infections has received substantial research,which includes pathogenderived resistance,introducing single or stacked transgene into crops,and RNA interference (Shepherd et al.,2009).However,these strategies have so far achieved limited success due to narrow availability and/or fitness costs on host plants.Therefore,it is urgent to develop novel and more efficient strategies to combat plant virus infections.Recent breakthrough of CRISPR/Cas-based DNA and RNA editing tools provides a promising direction for engineering artificial immunity to plant viruses.There are now increasing reports demonstrating that CRISPR/Cas systems can be harnessed to develop antiviral immunity in plants with high efficiency and broad specificity (Table S1 in Supporting Information).展开更多
Cutting-edge gene editing technologies enable broadened genomic alternations and accelerate opportunities to use these tools in biomedicine, agriculture, and animal model development. In this issue, Li et al.(2022) re...Cutting-edge gene editing technologies enable broadened genomic alternations and accelerate opportunities to use these tools in biomedicine, agriculture, and animal model development. In this issue, Li et al.(2022) reviews the tools of gene editing and highlights key technological developments and its broad applications in biomedicine, hoping to accelerate new discoveries and therapies in biomedicine.展开更多
This paper contains an error in Figure 2, where P171Y and P171A under treatment of pyroxsulam have a duplicated picture.This picture is for P171Y but not for P171A.We provide the correct picture for P171A as follows. ...This paper contains an error in Figure 2, where P171Y and P171A under treatment of pyroxsulam have a duplicated picture.This picture is for P171Y but not for P171A.We provide the correct picture for P171A as follows. This new picture does not affect the conclusion of this article.展开更多
Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance ...Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.展开更多
Dear Editor,Bread wheat(Triticum aestivum L.,2n=42,AABBDD),one of the three major food crops,is crucial for global food security and human sustenance.Significant efforts have been made in breeding programs to modify y...Dear Editor,Bread wheat(Triticum aestivum L.,2n=42,AABBDD),one of the three major food crops,is crucial for global food security and human sustenance.Significant efforts have been made in breeding programs to modify yield-related traits to enhance wheat productivity,with grain size being one of the critical targets for achieving this objective.展开更多
The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro...The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro cleavage assays and base editing analysis,we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1).We also show that these nicks are made on the target DNA strand.These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets.This system provides a novel tool for achieving trait stacking in plants.展开更多
The germplasm resource repository harbors an extensive collection of genetic variations,providing a crucial foundation for the survival and sustainable development of humankind.Throughout history,major agricultural br...The germplasm resource repository harbors an extensive collection of genetic variations,providing a crucial foundation for the survival and sustainable development of humankind.Throughout history,major agricultural breakthroughs have relied on safeguarding,exploring,and harnessing germplasm resources.However,the pursuit of high yields in modern agriculture has led to a continuous reduction in biodiversity,resulting in monocultures and an undesirable homogeneity of breeding materials.As a consequence,germplasm resources are facing the alarming prospect of accelerated loss leading to a decline in crop diversity.Furthermore,modern agricultural varieties encounter formidable challenges in terms of adapting to unfavorable growing conditions,such as environmental heterogeneity and the prevalence of pests and pathogens(Fig.1a).Enhancing the genetic variability of modern crops becomes paramount for fostering innovation within germplasm resources and ensuring food security.展开更多
In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively ...In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively inhibit the activity of Spy Cas9 both in bacterial and human cells.However,their molecular mechanisms remain elusive.Here,we report the cryo-electron microscopy structures of ternary complexes formed by AcrⅡA25.1 and AcrⅡA32 bound to Spy Cas9-sg RNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on Spy Cas9.We found that both AcrⅡA25.1 and AcrⅡA32 directly interact with the WED domain,where they spatially obstruct conformational changes of the WED and PI domains,thereby inhibiting Spy Cas9 from recognizing protospacer adjacent motif(PAM)and unwinding double-stranded DNA.In addition,they may inhibit nuclease activity by blocking the dynamic conformational changes of the Spy Cas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of Spy Cas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.展开更多
Hybrid breeding plays an important role in increasing crop yields by taking advantage of hybrid vigor,which helps crops grow with superior yields across various climates and contributes to ensuring secure food product...Hybrid breeding plays an important role in increasing crop yields by taking advantage of hybrid vigor,which helps crops grow with superior yields across various climates and contributes to ensuring secure food production.Hybrid seed production is relatively straightforward and simple in cross-pollinating plants such as maize but is difficult in self-pollinating plants such as rice,wheat,and soybean.Therefore,breeders have developed a three-line breeding strategy,utilizing a sterile line,a maintainer line,and a restorer line,for breeding hybrid rice(Figure 1A;Yuan,1966).展开更多
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020101 and XDA24020310)the National Natural Science Foundation of China(31672015,31788103)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003)。
文摘MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas(Cas12 a/Cpf1)family with a low level of homology to canonical Cas12 a nucleases.It has been publicly released as a royalty-free nuclease for both academic and commercial use.Here,we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences(YTTN)in plants.Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-Lb Cas12 a system.We develop two variants,MAD7-RR and MAD7-RVR that increase the target range of MAD7,as well as an M-AFID(a MAD7-APOBEC fusion-induced deletion)system that creates predictable deletions from 50-deaminated Cs to the MAD7-cleavage site.Moreover,we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs.Using the CRISPR-MAD7 system,we have obtained regenerated mutant rice and wheat plants with up to 65.6%efficiency.
基金supported by the National Natural Science Foundation of China(31788103 and 31971370)the National Key Research and Development Program(2022YFF1002802)the Ministry of Agriculture and Rural Affairs of China,and the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020102).
文摘CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.
基金This work was supported by the Strategic Priority Research Program of the CAS(Precision Seed Design and Breeding,XDA24020310 and XDA24020100)the National Natural Science Foundation of China(31830106 and 31872637)+2 种基金The Project for Extramural Scientists of the State Key Laboratory of Agro-Biotechnology(2021SKLAB6-7)Chinese Universities Scientific Fund(2021TC112)the Youth Innovation Promotion Association of the Chinese Academy of Sciences(2020000003).
文摘Genome editing provides novel strategies for improving plant traits,but relies on current genetic transformation and plant regeneration procedures,which can be inefficient.We have engineered a barley stripe mosaic virus(BSMV)-based sgRNA delivery vector(BSMV-sg)that is effective in performing heritable genome editing in Cas9-transgenic wheat plants.Mutated progenies were present in the next generation at frequencies ranging from 12.9%to 100%in three different wheat varieties,and 53.8%to 100%of mutants were virus-free.We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs.Furthermore,we devised a virus-induced transgene-free editing procedure(VITF-Edit)to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat.Our study provides a robust,convenient and tissue culture-free approach for genome editing in wheat through virus infection.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102 and XDA24010402)the National Natural Science Foundation of China (31788103 and 31971370)the Chinese Academy of Sciences (QYZDY-SSW-SMC030)
文摘Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.
基金supported by the Ministry of Agriculture of China(2016ZX08009-003)the Chinese Academy of Sciences(ZDRWZS-2019-2)the National Natural Science Foundation of China(31788103)。
文摘The seeds for genomics were sown with the development of DNA sequencing(Sanger et al., 1977), cultivated with each advance in molecular biology, and have since grown into one of the most important aspects of the life sciences. With the sequencing of the first draft human genome(Venter et al.,2001), the era of post-genomics began. Once one genome has been sequenced, it can be used as the reference for a certain species, and sequences from individuals can be mapped onto it to compare genetic variations among different lines.
基金supported by Key Projects in Science and Technology of Inner Mongolia (2021ZD0031)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA26030202)the Youth Innovation Promotion Association,CAS (2022096)。
文摘The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops.Here,we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)genome editing in a perennial,stress-tolerant forage grass,sheepgrass(Leymus chinensis).By screening for active single-guide RNAs(sg RNAs),accessions that regenerate well,suitable Agrobacterium strains,and optimal culture media,and co-expressing the morphogenic factor Ta WOX5,we achieved 11%transformation and 5.83%editing efficiency in sheepgrass.Knocking out Teosinte Branched1(TB1)significantly increased tiller number and biomass.This study opens avenues for studying gene function and breeding in sheepgrass.
基金supported by the National Key Research and Development Program of China (2016YFD0101800)the National Natural Science Foundation of China (91635301)+1 种基金the Zhejiang Provincial Natural Science Foundation of China (LZ14C130003)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences
文摘Dear Editor,The CRISPR-Cas9（clustered regularly interspaced short palindromic repeats/Cas9）system has been widely used for a variety of applications,including targeted gene knockout,gene insertion,gene replacement and base editing.Despite its wide use,the genome editing using CRISPR-Cas9 is performed almost exclusively at sites containing canonical NGG protospacer adjacent motifs（PAMs）.To overcome the PAM constraint of the CRISPR-Cas9 system,many attempts have been made to develop various Cas9 orthologs and v variants with altered PAM specificities （Kleinstiver et al., 2015; Hu et al., 2016）.
文摘Ongoing advances in genomics and genetics have led researchers to highlight the significant role of large DNA structural variants(SVs)in genetic diversity.SVs are genomic sequence variants of various types,including insertions,deletions,inversions,translocations,and copy number variants(CNVs or duplications).These SVs,which are usually of 1 kb or more,can have profound impacts at both the individual and species level.They are also often linked to the initiation and progression of complex genetic diseases(Stankiewicz and Lupski,2010).
基金supported by grants from the National Key Research and Development Program of China (2016YFD0101804)the National Natural Science Foundation of China (31788103 and 31420103912)as well as the Chinese Academy of Sciences (QYZDY-SSWSMC030 and GJHZ1602)
文摘Precursor-mRNAs(pre-mRNA) can be processed into one or more mature m RNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intronmediated enhancement(IME), whereas alternative splicing(AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5′ splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB 1.1(encoding a type 2 C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31 A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly,altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.
基金supported by grants from the National Key R&D Program of China(2018YFA0900600)the National Natural Science Foundation of China(31900301,31872933 and 31971370)the Chinese Academy of Sciences(QYZDY-SSW-SMC030).
文摘Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.
基金We gratefully ack no wledge funding from the NSFC for the Basic Scie nee Center Program(31788103)the National Natural Science Foundation of China(31970529)the China Postdoctoral Science Foundation(2019M650892).
文摘Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.
基金supported by grants from the National Natural Science Foundation of China (31788103)the National Key Research and Development Program of China (2016YFD0101804)
文摘Virus infection poses a constant threat on global crop productivity.It is estimated that virus diseases account for at least 10% of crop losses worldwide.Development of resistant crops using naturally evolved resistance genes has been a main measure for controlling plant virus epidemics.However,isolation of plant resistance genes is costly and time-consuming.Furthermore,most plant resistance genes are pathogen race-specific,with very few of them being broadly specific (Langner et al.,2018).Consequently,artificial immunity with engineered resistance to virus infections has received substantial research,which includes pathogenderived resistance,introducing single or stacked transgene into crops,and RNA interference (Shepherd et al.,2009).However,these strategies have so far achieved limited success due to narrow availability and/or fitness costs on host plants.Therefore,it is urgent to develop novel and more efficient strategies to combat plant virus infections.Recent breakthrough of CRISPR/Cas-based DNA and RNA editing tools provides a promising direction for engineering artificial immunity to plant viruses.There are now increasing reports demonstrating that CRISPR/Cas systems can be harnessed to develop antiviral immunity in plants with high efficiency and broad specificity (Table S1 in Supporting Information).
基金supported by the National Natural Science Foundation of China (Grant No. 41988101)K.C.Wong Education Foundation
文摘Cutting-edge gene editing technologies enable broadened genomic alternations and accelerate opportunities to use these tools in biomedicine, agriculture, and animal model development. In this issue, Li et al.(2022) reviews the tools of gene editing and highlights key technological developments and its broad applications in biomedicine, hoping to accelerate new discoveries and therapies in biomedicine.
文摘This paper contains an error in Figure 2, where P171Y and P171A under treatment of pyroxsulam have a duplicated picture.This picture is for P171Y but not for P171A.We provide the correct picture for P171A as follows. This new picture does not affect the conclusion of this article.
基金supported by grants from the National Natural Science Foundation of China(31788103 and 31971370)the National Transgenic Science and Technology Program(2016ZX08010002 and 2018ZX0801002B)。
文摘Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.
基金supported by grants from the Key Research and Development Project of Shandong Province (the Agricultural Variety Improvement Project,2023LZGC002)the National Key R&D Program of China (2023YFF1001600)+1 种基金the National Natural Science Foundation of China (32388201)the Ministry of Agriculture and Rural Affairs of China and the New Cornerstone Science Foundation。
文摘Dear Editor,Bread wheat(Triticum aestivum L.,2n=42,AABBDD),one of the three major food crops,is crucial for global food security and human sustenance.Significant efforts have been made in breeding programs to modify yield-related traits to enhance wheat productivity,with grain size being one of the critical targets for achieving this objective.
基金supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020102)the National Transgenic Science and Technology Program(2018ZX0801002B)+2 种基金the National Natural Science Foundation of China(31788103 and 31971370)the Chinese Academy of Sciences(QYZDY-SSW-SMC030)the National Key R&D Program of China(2018YFA0900600,2016YFD0100102-11,and 2016YFD0100605)。
文摘The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro cleavage assays and base editing analysis,we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1).We also show that these nicks are made on the target DNA strand.These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets.This system provides a novel tool for achieving trait stacking in plants.
基金supported by the National Natural Science Foundation of China(32025028,U20A2030,32188102,and 32388201)the National Key Research and Development Program of China(2022YFF1003304)+1 种基金the earmarked fund for China Agriculture Research System(CARS)the Hainan Yazhou Bay Seed Laboratory(B21HJ0215)。
文摘The germplasm resource repository harbors an extensive collection of genetic variations,providing a crucial foundation for the survival and sustainable development of humankind.Throughout history,major agricultural breakthroughs have relied on safeguarding,exploring,and harnessing germplasm resources.However,the pursuit of high yields in modern agriculture has led to a continuous reduction in biodiversity,resulting in monocultures and an undesirable homogeneity of breeding materials.As a consequence,germplasm resources are facing the alarming prospect of accelerated loss leading to a decline in crop diversity.Furthermore,modern agricultural varieties encounter formidable challenges in terms of adapting to unfavorable growing conditions,such as environmental heterogeneity and the prevalence of pests and pathogens(Fig.1a).Enhancing the genetic variability of modern crops becomes paramount for fostering innovation within germplasm resources and ensuring food security.
基金supported by the National Key Research and Development Program of China(2023YFF1000200)the National Natural Science Foundation of China(U21A20276)+1 种基金the Tencent Foundation through the XPLORER PRIZE and the New Cornerstone Science Foundation to Z.H.,Heilongjiang Touyan Team(HITTY-20190034 to Z.H.)Natural Science Foundation of Heilongjiang Province of China(YQ2023C032 to Y.Z.)。
文摘In the ongoing arms race between bacteria and bacteriophages,bacteriophages have evolved anti-CRISPR proteins to counteract bacterial CRISPR-Cas systems.Recently,AcrⅡA25.1 and AcrⅡA32 have been found to effectively inhibit the activity of Spy Cas9 both in bacterial and human cells.However,their molecular mechanisms remain elusive.Here,we report the cryo-electron microscopy structures of ternary complexes formed by AcrⅡA25.1 and AcrⅡA32 bound to Spy Cas9-sg RNA.Using structural analysis and biochemical experiments,we revealed that AcrⅡA25.1 and AcrⅡA32 recognize a novel,previously-unidentified anti-CRISPR binding site on Spy Cas9.We found that both AcrⅡA25.1 and AcrⅡA32 directly interact with the WED domain,where they spatially obstruct conformational changes of the WED and PI domains,thereby inhibiting Spy Cas9 from recognizing protospacer adjacent motif(PAM)and unwinding double-stranded DNA.In addition,they may inhibit nuclease activity by blocking the dynamic conformational changes of the Spy Cas9 surveillance complex.In summary,our data elucidate the inhibition mechanisms of two new anti-CRISPR proteins,provide new strategies for the modulation of Spy Cas9 activity,and expand our understanding of the diversity of anti-CRISPR protein inhibition mechanisms.
基金supported by the National Natural Science Foundation of China(32388201)the National Key Research and Development Program(2022YFF1002802)the Ministry of Agriculture and Rural Affairs of China,the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020102) and the New Cornerstone Science Foundation through the New Cornerstone Investigator Program.
文摘Hybrid breeding plays an important role in increasing crop yields by taking advantage of hybrid vigor,which helps crops grow with superior yields across various climates and contributes to ensuring secure food production.Hybrid seed production is relatively straightforward and simple in cross-pollinating plants such as maize but is difficult in self-pollinating plants such as rice,wheat,and soybean.Therefore,breeders have developed a three-line breeding strategy,utilizing a sterile line,a maintainer line,and a restorer line,for breeding hybrid rice(Figure 1A;Yuan,1966).
