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Rock paper scissors: CRISPR/Cas9-mediated interference with geminiviruses in plants

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Science China(Life Sciences) 2019年第10期62卷 1389-1391页

作者： Xiuling Yang huanbin zhou Xueping zhou State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural Sciences Beijing 100193China State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

Geminiviruses are a group of circular single-stranded DNA viruses that constitute the largest family of plant viruses. Many diseases resulting from geminivirus infections, such as maize streak disease, cassava mosaic disease, tomato yellow leaf curl disease and cotton leaf curl diseases, cause significant problems in terms of economic losses, posing a serious threat to global crop productivity (Hanley-Bowdoin et al., 2013;Yang et al., 2019). As obligate intracellular parasites, geminiviruses cannot be controlled chemically. Conventional strategies to control geminiviruses rely heavily on the application of pesticides to limit insect vector populations, but it causes environmental concerns. To date, developing novel crop germplasms with durable disease resistance to geminiviruses is still the best strategy to manage viral infections due to its environmental friendliness. However, traditional breeding programs are time-intensive and labor-consuming processes, and transgene-based modern approaches by overexpressing the geminiviral proteins (e.g., the truncated form of the replication initiator protein) failed to confer resistance to geminiviruses due to activated virusinduced transgene silencing.

关键词： mosaic disease insect vector disease resistance

Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

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The Crop Journal 2023年第2期11卷 661-665页

作者： Bin Ren Yongjie Kuang Ziyan Xu Xuemei Wu Dawei Zhang Fang Yan Xiangju Li Xueping zhou Guirong Wang huanbin zhou Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 440307GuangdongChina State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Scientific Observing and Experimental Station of Crop Pests in Guilin Ministry of Agriculture and Rural AffairsGuilin 541399GuangxiChina State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058ZhejiangChina Ministry of Education Key Laboratory of Bio-Resource and Eco-Environment College of Life SciencesSichuan UniversityChengdu 610065SichuanChina

Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

关键词： Base editing Gene evolution Glutamine synthetase Glufosinate tolerance Rice

Adenine base editor incorporating the N-methylpurine DNA glycosylase MPGv3 enables efficient A-to-K base editing in rice

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Plant Communications 2023年第6期4卷 230-233页

作者： Xuemei Wu Bin Ren Lang Liu Shengqun Qiu Xin’ge Li Peijing Li Fang Yan Honghui Lin Xueping zhou Dawei Zhang huanbin zhou Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment College of Life ScienceState Key Laboratory of Hydraulics and Mountain River EngineeringSichuan UniversityChengdu 610064China State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Scientific Observing and Experimental Station of Crop Pests in Guilin Ministry of Agriculture and Rural AffairsGuilin 541399China Department of Plant Pathology China Agricultural UniversityBeijing 100193China State Key Laboratory of North China Crop Improvement and Regulation College of Plant ProtectionHebei Agricultural UniversityBaoding 071000China School of Life Science and Technology Hubei Engineering UniversityXiaogan 432000China State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China Hainan Yazhou Bay Seed Lab/National Nanfan Research Institute(Sanya) Chinese Academy of Agricultural SciencesSanya 572024China

Dear Editor,Crop genetic diversity and elite agronomic traits are mainly caused by genetic variants,approximately half of which are single-nucleotide polymorphisms.Precise nucleotide substitution through CRISPR–Cas-mediated base editors has been widely used to correct defective alleles and create novel alleles by artificial evolution for rapid crop genetic improvement.Since 2017,cytosine base editors(CBEs)and adenine base editors(ABEs)have been successively developed in many plant species and have been continuously optimized to generate highly efficient C-to-T and A-to-G transitions,as well as by-product C-to-A/G conversion with low efficiency(Ren et al.,2018;Yan et al.,2021).

关键词： elite base editor

Co-evolved plant and blast fungus ascorbate oxidases orchestrate the redox state of host apoplast to modulate rice immunity

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Molecular Plant 2022年第8期15卷 1347-1366页

作者： Jiexiong Hu Muxing Liu Ao Zhang Ying Dai Weizhong Chen Fang Chen Wenya Wang Danyu Shen Mary Jeanie Telebanco-Yanoria Bin Ren Haifeng Zhang huanbin zhou Bo zhou Ping Wang Zhengguang Zhang Department of Plant Pathology College of Plant ProtectionNanjing Agricultural Universityand Key Laboratory of Integrated Management of Crop Diseases and PestsMinistryof EducationNanjingChina The Key Laboratory of Plant Immunity Nanjing Agricultural UniversityNanjingChina Genetics and Biotechnology Division International Rice Research InstituteLos BanosLaguna 4031Philippines State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Department of Microbiology Immunologyand ParasitologyLouisiana State University Health Sciences CenterNew OrleansLAUSA

Apoplastic ascorbate oxidases(AOs)play a critical role in reactive oxygen species(RoS)-mediated innate host immunity by regulating the apoplast redox state.To date,little is known about how apoplastic effectors of the riceblast fungus Magnaportheoryzaemodulate the apoplast redox state of rice to subvert plant immunity.In this study,we demonstrated that M.oryzae MoAo1 is an Ao that plays a role in virulence by modulating the apoplast redox status of rice cells.We showed that MoAo1 inhibits the activity of rice OsAO3and OsAO4,which also regulate the apoplast redox status and plant immunity.In addition,we found that MoAo1,OsAO3,andOsAO4 allexhibit polymorphic variations whosevaried interactions orchestrate pathogen virulence and rice immunity.Taken together,our results reveal a critical role for extracellular redox enzymes during rice blast infection and shed light on the importance of the apoplast redox state anditsregulation inplant-pathogeninteractions.

关键词： apoplast redox state RoS ascorbate oxidase polymorphism rice blast host immunity

Conferring Resistance to Plant RNA Viruses with the CRISPR/CasRx System

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Virologica Sinica 2021年第4期36卷 814-817页

作者： Yongsen Cao huanbin zhou Xueping zhou Fangfang Li State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

Dear Editor,Clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is an adaptive immune system discovered in prokaryotic bacteria or archaea that can fend off invading nucleic acids.Because of its simplicity,high efficiency and versatility,CRISPR/Cas system-mediated genome editing has been widely applied in plant research and agricultural production.

关键词： CRISPR/Cas system production.

Base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms

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Molecular Plant 2020年第4期13卷 565-572页

作者： Yongjie Kuang Shaofang Li Bin Ren Fang Yan Carl Spetz Xiangju Li Xueping zhou huanbin zhou State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China College of Biological Sciences China Agricultural UniversityBeijing 100193China Scientific Observing and Experimental Station of Crop Pests in Guilin Ministry of Agriculture and Rural AffairsGuilin 541399China Norwegian Institute of Bioeconomy Research 1432 AasNorway State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

关键词： CRISPR base editor gene evolution OsALS1 herbicide resistance Otyza sativa L

CRISPR/Sc^(++)-mediated genome editing in rice

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Journal of Integrative Plant Biology 2021年第9期63卷 1606-1610页

作者： Guigen Ma Yongjie Kuang Zhenwan Lu Xueqi Li Ziyan Xu Bin Ren Xueping zhou huanbin zhou State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Scientific Observing and Experimental Station of Crop Pests in Guilin Ministry of Agriculture and Rural AffairsGuilin 541399China State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

Streptococcus canis Cas9(ScCas9)is an RNAguided endonuclease with NNG protospacer adjacent motif(PAM)specificity whose genomeediting activity in rice is locus-dependent.Here we investigated the performance of a ScCas9 variant named Sc^(++)at different NNG PAM sites in the rice genome;Sc^(++)harbors a T1227K mutation and the substitution of a positively charged loop(residues 367-376).Sc^(++)nuclease achieved broader genome editing compared to the original ScCas9,and its nickase improved targeted base editing in transgenic rice plants.Using the high-efficiency adenine base editor rBE73b,we generated many new OsGS1 alleles suitable for screening of rice germplasm for potential herbicide resistance in the future.The CRISPR/Sc^(++)system expands the genomeediting toolkit for rice.

关键词： base editing genome editing rice Sc^(++)

High-efficiency and multiplex adenine base editing in plants using new TadA variants

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Molecular Plant 2021年第5期14卷 722-731页

作者： Daqi Yan Bin Ren Lang Liu Fang Yan Shaofang Li Guirong Wang Wenxian Sun Xueping zhou huanbin zhou State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Department of Plant Pathology China Agricultural UniversityBeijing 100193China Scientific Observing and Experimental Station of Crop Pests in Guilin Ministry of Agriculture and Rural AffairsGuilin 541399China Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518120China College of Biological Sciences China Agricultural UniversityBeijing 100193China College of Plant Protection Jilin Agricultural UniversityChangchun 130118China State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

Recently reported adenine base editors(ABEs)exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding.However,editing efficiency varies significantly among different target sites.Here,we investigated the activities of three evolved E.coli adenosine deaminase TadA variants(TadA8e,TadA8.17,and TadA8.20)side-by-side in transgenic rice.We found that TadA8e outperforms TadA8.17 and TadA8.20,and induces efficient A-to-G conversion at all tested sites in the rice genome,including those that were un-editable by ABE7.10 in our previous experiments.Furthermore,V82S/Q154R mutations were incorporated into TadA8e,resulting in a new variant that we named TadA9.Our data show that TadA9 is broadly compatible with CRISPR/SpCas9,CRISPR/SpCas9-NG,and CRISPR/SpRY,as well as CRISPR/ScCas9 nickase systems,achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e.Finally,TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in.weed control.Collectively,we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome.Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.

关键词： CRISPR TadA variant adenine base editing Oryza sativa L.

Cas9-NG Greatly Expands the Targeting Scope of the Genome-Editing Toolkit by Recognizing NG and Other Atypical PAMs in Rice

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Molecular Plant 2019年第7期12卷 1015-1026页

作者： Bin Ren Lang Liu Shaofang Li Yongjie Kuang Jingwen Wang Dawei Zhang Xueping zhou Honghui Lin huanbin zhou Ministry of Education Key Laboratory of Bio-Resource and Eco-Environment College of Life Sciences Sichuan University Chengdu 610065 China State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100193 China State Key Laboratory of Plant Physiology and Biochemistry College of Biological Sciences China Agricultural University Beijing 100193 China State Key Laboratory of Rice Biology Institute of Biotechnology Zhejiang University Hangzhou 310058 China

CRISPR technologies enabling precise genome manipulation are valuable for gene function studies and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM)y such as NGG and TTN, for Cas protein recognition restricts the selection of targetable genomic loci in practical applications of CRISPR technologies. Recently Cas9-NG, which recognizes a minimal NG PAM, was reported to expand the targeting space of genome editing in human cells, but it remains unclear whether this Cas9 variant can be used in plants. In this study, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice. We found that Cas9-NG edits all NGG, NGA, NGT, and NGC sites with impaired activity, while the gene-edited plants were dominated by monoallelic mutations. Cas9-NG-engineered base editors were then developed and used to generate O s B Z R I gainof- function plants that can not be created by other available Cas9-engineered base editors. Moreover, we showed that a Cas9-NG-based transcriptional activator efficiently upregulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, and NCG apart from NG PAM. Together, these findings demonstrate that Cas9-NG can greatly expand the targeting scope of genome-editing tools, showing great potential for targeted genome editing, base editing, and genome regulation in plants.

关键词： CRISPR Cas9-NG PAM gene editing base editing Oryza sativa L