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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

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

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)

Leaching kinetics of fluorine during the aluminum removal from spent li-ion battery cathode materials

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Journal of Environmental Sciences 2024年第4期138卷 312-325页

作者： Shengjie li Jianxin Zhu Research Center for Eco-Environmental Sciences Chinese Academy of SciencesBeijing 100085China University of Chinese Academy of Sciences Beijing 100049China

The high content of aluminum(Al)impurity in the recycled cathode powder seriously affects the extraction efficiency of Nickel,Cobalt,Manganese,and lithium resources and the actual commercial value of recycled materials,so Al removal is crucially important to conform to the industrial standard of spent li-ion battery cathode materials.In this work,we systematically investigated the leaching process and optimum conditions associated with Al removal from the cathode powder materials collected in a wet cathode-powder peeling and recycling production line of spent li-ion batteries(liBs).Moreover,we specifically studied the leaching of fluorine(F)synergistically happened along with the removal process of Al,which was not concerned about in other studies,but one of the key factors affecting pollution prevention in the recovery process.The mechanism of the whole process including the leaching of Al and F from the cathode powder was indicated by using NMR,FTIR,and XPS,and a defluoridation process was preliminarily investigated in this study.The leaching kinetics of Al could be successfully described by the shrinking core model,controlled by the diffusion process and the activation energy was 11.14 kJ/mol.While,the leaching of F was attributed to the dissolution of liPF6and decomposition of PVDF,and the kinetics associated was described by Avrami model.The interaction of Al and F is advantageous to realize the defluoridation to some degree.It is expected that our investigation will provide theoretical support for the large-scale recycling of spent liBs.

关键词： Al removal F leaching Cathode powder Heating alkali leaching Leaching kinetics

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

Additives strategies to improving cycling stability of li metal batteries by modifying electrode electrolyte interphases

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Progress in Natural Science:Materials International 2024年第2期34卷 338-344页

作者： Yuan li Pengfei Huang Chunyu Cui Xiaoyuan Ma li Chen Zekun li linhai li Yuhong Tian School of Biological and Chemical Engineering Guangxi University of Science and Technology School of College of Chemistry and Chemical Engineering Hunan University Isky Future Energy Institute

li metal batteries（LMBs） have attracted much attention due to the ultra-high theoretical capacity of the li metal anode（LMA）.However,the uncontrollable dendrites growth and low coulombic efficiency hinder their practical application.Here,we explore 3,5-difluoropyridine（2F-BD） as a novel electrolyte additive to enable highperformance LMBs.The 2F-BD additive participates in and modifies the solvation structure of li ions by reducing the coordinated number of PF6-at the electrode surface,leading to the formation of a liF and li3N-rich solid electrolyte interphase（SEI）.The liF component increases the robustness of SEI and suppresses the formation of li dendrites,while the li3N component facilitates the transportation and reaction kinetics of li ions.As a result,the li‖li symmetric cell presents a stable cycling performance of up to 500 h at a current density of 1 mA cm-2.After coupling with liFePO4cathode,the obtained full cell achieves high specific capacities of 106.06 mA h g-1and 84.98 mA h g-1at 2.55 mA cm-2and 5.10 mA cm-2,respectively,and maintains a high capacity retention of84.0% after 1000 cycles at 0.13 mA cm-2,with an average cycling CE of 99.58%.

关键词： Electrolyte additive li dendrites li metal batteries liF and li3N-Rich electrode-electrolyte interphase liFePO4 cathode

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.)

Base editing in plants: Current status and challenges

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The Crop Journal 2020年第3期8卷 384-395页

作者： Sutar Suhas Bharat shaoya li Jingying li Lei Yan Lanqin Xia Institute of Crop Sciences Chinese Academy of Agricultural SciencesBeijing 100081China

Genome editing technologies have revolutionized the field of plant science by enabling targeted modification of plant genomes and are emerging as powerful tools for both plant gene functional analyses and crop improvement. Although homology-directed repair(HDR)is a feasible approach to achieve precise gene replacement and base substitution in some plant species, the dominance of the non-homologous end joining pathway and low efficiency of HDR in plant cells have limited its application. Base editing has emerged as an alternative tool to HDR-mediated replacement, facilitating precise editing of plant genome by converting one single base to another in a programmable manner without a doublestranded break and a donor repair template. In this review, we summarize the latest developments in base-editing technologies as well as their underlying mechanisms. We review current applications of these technologies in plant species. Finally, we address the challenges and future perspectives of this emerging technology in plants.

关键词： Base editing in plants Current status and challenges