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Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance

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植物学报:英文版 2023年第9期65卷 2194-2203页

作者： Chen Zhang Xue Zhong Shaoya Li Lei Yan Jingying Li Yubing He Yong Lin Yangjun Zhang lanqin xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China CAAS/Hainan Yazhou Bay Seed Laboratory National Nanfan Research Institute(Sanya)Sanya 572024China Key Laboratory of Gene Editing Technologies(Hainan) Ministry of Agricultural and Rural AffairsSanya 572024China Beijing Dabeinong Technology Group Co. LtdBeijing 10080China

Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn(D213N)mutation(OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.

关键词： artificial evolution dual cytosine and adenine base editor nCas9‐NG rice(Oryza sativa L) near‐saturated mutagenesis

Precise gene replacement in plants through CRISPR/Cas genome editing technology:current status and future perspectives

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aBIOTECH 2020年第1期1卷 58-73页

作者： Shaoya Li lanqin xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China

CRISPR/Cas,as a simple,versatile,robust and cost-effective system for genome manipulation,has dominated the genome editing field over the past few years.The application of CRISPR/Cas in crop improvement is particularly important in the context of global climate change,as well as diverse agricultural,environmental and ecological challenges.Various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci,transcriptome regulation and epigenome editing,base editing,and precise targeted gene/allele replacement or tagging in plants.In particular,precise replacement of an existing allele with an elite allele in a commercial variety through homology-directed repair(HDR)is a holy grail in genome editing for crop improvement as it has been very difficult,laborious and time-consuming to introgress the elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop breeding practice.However,it still remains very challenging in crop plants.This review intends to provide an informative summary of the latest development and breakthroughs in gene replacement using CRISPR/Cas technology,with a focus on achievements,potential mechanisms and future perspectives in plant biological science as well as crop improvement.

关键词： CRISPR/Cas Gene targeting(GT) Gene/allele replacement Genome editing Homology-directed repair(HDR)

Multiplex precision gene editing by a surrogate prime editor in rice

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Molecular Plant 2022年第7期15卷 1077-1080页

作者： Huiyuan Li Ziwei Zhu Shaoya Li Jingying Li Lei Yan Chen Zhang Youzhi Ma lanqin xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Be11ing 100081China Hainan Yazhou Bay Seed Laboratory/National Nanfan Research Institute(Sanya) Chinese Academy of Agricultural SciencesSanyaHainan Province 572024China

Dear Editor,Development of a multiplex precision gene editing system is highly desirable for pyramiding beneficial alleles in crop improve-ment.Prime editing is a newly developed genome-editing tool that can precisely enable the installation of all 12 nucleotide sub-stitutions,short insertions,and deletions without exogenous DNA donor repair template and double-strand breaks(Anzalone et al.,2019).Among the prime editing systems,prime editor 3(PE3),which consists of a prime editing guide RNA(pegRNA)and an additional nicking single guide RNA(sgRNA)to nick the non-edited strand,enhances editing efficiency.

关键词： editor editing template

Engineering a plant A-to-K base editor with improved performance by fusion with a transactivation module

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Plant Communications 2023年第6期4卷 225-229页

作者： Yucai Li Shaoya Li Chenfei Li Chen Zhang Lei Yan Jingying Li Yubing He Yan Guo Yong Lin Yangjun Zhang lanqin xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China Hainan Yazhou Bay Seed Laboratory/National Nanfan Research Institute(Sanya) CAASSanyaHainan Province 572024China State Key Laboratory of Plant Physiology and Biochemistry College of Biological SciencesChina Agricultural UniversityBeijing 100193China Beijing Dabeinong Technology Group Co. Ltd.Beijing 10080China

Dear Editor,Base editors(BEs)are promising tools that enable single-nucleotide substitutions in specific target genes,generating loss-offunction or gain-of-function mutations,which can greatly accelerate plant functional genomics and crop improvement(Ren et al.,2018;Zeng et al.,2020;Xu et al.,2021;Yan et al.,2021;Li et al.,2023).To date,three major classes of BEs have been engineered:cytosine BEs(CBEs)for C-to-T transitions(Komor et al.,2016),adenine BEs(ABEs)for A-to-G transitions(Gaudelli et al.,2017).

关键词： CBE editor base

The power and versatility of genome editing tools in crop improvement

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Journal of Integrative Plant Biology 2021年第9期63卷 1591-1594页

作者： lanqin xia Kejian Wang Jian-Kang Zhu Special Issue Editors 1.Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China State Key Laboratory of Rice Biology China National Rice Research InstituteHangzhou 310006China Shanghai Center for Plant Stress Biology Center for Excellence in Molecular Plant SciencesChinese Academy of SciencesShanghai 200032China

Increasing world population,global climate change,decreased farmland,environmental pollution and ecological deterioration represent unprecedent challenges for crop production to ensure global food security(Hickey et al.,2019;Li et al.,2021 a).It is estimated that by the year 2050,50% more food is needed to feed the increasing population(Bailey-Serres et al.,2019).Thus,it is urgent to boost crop production by using cutting-edge technologies.

关键词： farmland pollution boost

Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9

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Journal of Integrative Plant Biology 2021年第9期63卷 1649-1663页

作者： Jiahui Zhang Huating Zhang Shaoya Li Jingying Li Lei Yan lanqin xia Institute of Crop Sciences Chinese Academy of Agricultural SciencesBeijing 100081China School of Agricultural Sciences Zhengzhou UniversityZhengzhou 450001China

Wheat(Triticum aestivum L.)is a staple food crop consumed by more than 30%of world population.Nitrogen(N)fertilizer has been applied broadly in agriculture practice to improve wheat yield to meet the growing demands for food production.However,undue N fertilizer application and the low N use efficiency(NUE)of modern wheat varieties are aggravating environmental pollution and ecological deterioration.Under nitrogen-limiting conditions,the rice(Oryza sativa)abnormal cytokinin response1 repressor1(are1)mutant exhibits increased NUE,delayed senescence and consequently,increased grain yield.However,the function of ARE1 ortholog in wheat remains unknown.Here,we isolated and characterized three TaARE1 homoeologs from the elite Chinese winter wheat cultivar ZhengMai 7698.We then used CRISPR/Cas9-mediated targeted mutagenesis to generate a series of transgene-free mutant lines either with partial or triple-null taare1 alleles.All transgene-free mutant lines showed enhanced tolerance to N starvation,and showed delayed senescence and increased grain yield in field conditions.In particular,the AABBdd and aabbDD mutant lines exhibited delayed senescence and significantly increased grain yield without growth defects compared to the wild-type control.Together,our results underscore the potential to manipulate ARE1 orthologs through gene editing for breeding of high-yield wheat as well as other cereal crops with improved NUE.

关键词： CRISPR Increasing yield potential through manipulating of an ARE1 ortholog related to nitrogen use efficiency in wheat by CRISPR/Cas9

Toward Precision Genome Editing in Crop Plants

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Molecular Plant 2020年第6期13卷 811-813页

作者： Jingying Li Huiyuan Li Jilin Chen Lei Yan lanqin xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China

Precision genome editing through homology-directed repair(HDR)in plants remains very challenging due to the innately low occurrence of HDR and obstacles in the delivery of sufficient donor repair template(DRT)into plant cells.For single-base substitution,cytidine or adenine base editors have been used successfully in crop plants in recent years.However,base editing is constrained by the distance between the targeted base and PAM motif and cannot install predefined transversion mutations or insertions and deletions(indels).Here,we briefly summarize the strategies reported so far for precision genome editing in plants.Based on the recently developed prime editing strategy,which enables gene replacement without double-strand breaks(DSBs)or DRT in both human cells and plants,we provide our perspectives on exploiting diverse strategies to achieve precise targeted gene/allele replacement toward crop improvement and resilience for sustainable agricultural development.

关键词： base install template

Precise Modifications of Both Exogenous and Endogenous Genes in Rice by Prime Editing

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Molecular Plant 2020年第5期13卷 671-674页

作者： Huiyuan Li Jingying Li Jilin Chen Lei Yan lanqin xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China

Dear Editor,Harnessing genetic diversity and the introduction of elite alleles from wild relatives or landraces into commercial cultivars has been a major goal in crop breeding programs.Precise modification of the plant genome through clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein(Cas)(CRISPR/Cas)-mediated homology-directed repair(HDR)offers a great promise to introduce elite alleles from wild relatives or landraces into commercialized cultivars in the short term.

关键词： cultivar elite CRISPR/Cas

Present and future prospects for wheat improvement through genome editing and advanced technologies

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Plant Communications 2021年第4期2卷 100-115页

作者： Shaoya Li Chen Zhang Jingying Li Lei Yan Ning Wang lanqin xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China

Wheat(Triticum aestivum,2n=6x=42,AABBDD)is one of the most important staple food crops in the world.Despite the fact that wheat production has significantly increased over the past decades,future wheat production will face unprecedented challenges from global climate change,increasing world population,and water shortages in arid and semi-arid lands.Furthermore,excessive applications of diverse fertilizers and pesticides are exacerbating environmental pollution and ecological deterioration.To ensure global food and ecosystem security,it is essential to enhance the resilience of wheat production while minimizing environmental pollution through the use of cutting-edge technologies.However,the hexaploid genome and gene redundancy complicate advances in genetic research and precision gene modifications for wheat improvement,thus impeding the breeding of elite wheat cultivars.In this review,we first introduce state-of-the-art genome-editing technologies in crop plants,especially wheat,for both functional genomics and genetic improvement.We then outline applications of other technologies,such as GWAS,high-throughput genotyping and phenotyping,speed breeding,and synthetic biology,in wheat.Finally,we discuss existing challenges in wheat genome editing and future prospects for precision gene modifications using advanced genome-editing technologies.We conclude that the combination of genome editing and other molecular breeding strategies will greatly facilitate genetic improvement ofwheat for sustainable global production.

关键词： CRISPR/Cas genome editing GWAS speed breeding wheat(Triticum aestivum L.)