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High-throughput Genome editing in rice with a virus-based surrogate system

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Journal of Integrative Plant Biology 2023年第3期65卷 646-655页

作者： Yifu Tian Dating Zhong Xinbo Li Rundong Shen Han Han Yuqin Dai Qi Yao Xuening Zhang Qi Deng Xuesong Cao Jian-Kang Zhu Yuming Lu Shanghai Center for Plant Stress Biology Center for Excellence in Molecular Plant SciencesChinese Academy of SciencesShanghai 201602China Center for Advanced Bioindustry Technologies Institute of Crop SciencesChinese Academy of Agricultural SciencesBeiing 100081China Hainan Yazhou Bay Seed Lab Sanya 572024China Shanghai Collaborative Innovation Center of Agri Seeds Joint Center for Single Cell BiologySchool of Agriculture and BiologyShanghai Jiao Tong UniversityShanghai 200240China Institute of Advanced Biotechnology School of Life SciencesSouthern University of Science and TechnologyShenzhen 518055China

With the widespread use of clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas) technologies in plants, large-scale Genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate(WDV-surrogate), to facilitate high-throughput Genome editing.WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene(HygM) for surrogate selection;the other is a WDV-based construct expressing two single guide RNAs(sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T_0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency(66.8% vs. 90.1%), plantlet regeneration efficiency(2.31-fold increase), and numbers of homozygous-edited plants(36.3%vs. 70.7%). Large-scale editing using pooled sg RNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDVGate on sequence knock-in for protein tagging.By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.

关键词： Genome editing high-throughput protein tagging rice

A novel non-invasive identification of Genome editing mutants from insect exuviae

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Insect Science 2022年第1期29卷 21-32页

作者： Xiao-Xiao Wang Jing Li Tong-Xin Wang Yi-Nuo Yang Hai-Kang Zhang Meng Zhou Le Kang Li-Ya Wei College of Life Sciences Institute of Life Sciences and Green DevelopmentHebei UniversityBaodingHebei Province071002China State Key Laboratory of Integrated Management of Pest Insects and Rodents Institute of ZoologyChinese Academy of SciencesBeijing100101China

With the wide application of Genome editing in insects, a simple and efficient identification method is urgently needed to meet the increasing demand for mutation detection. Here, taking migratory locusts as a model system, we developed a non-invasive method to accurately identify genome-edited mutants by using DNA from insect exuviae. We compared the quantity and quality of genomic DNA from exuviae in five instar hoppers and found that the 1st instar exuviae had the highest DNA yield and content, while the 3rd instar exuviae had the best quality. Consensus genotypes were identified from genomic DNA of hoppers at different developmental stages in the same individuals. Moreover, we demonstrated that the amplification products from DNA extracted from locust exuviae are the consensus sequences with those from the hemolymph and foreleg pre-tarsus. Therefore, non-invasive samples provide the same genotyping results as minimally invasive and invasive samples of the same individuals. Furthermore, this identification method that uses genomic DNA from exuviae can be used for early screening of positive genome-edited individuals in each generation for adult crossing. In our study, the non-invasive identification method was not only simpler and provided results earlier than existing methods, but also had a better reproducibility and accuracy. This non-invasive identification approach using genomic DNA from exuviae can be adapted to meet the growing demand for genetic analysis and will find wide application in insect Genome editing research.

关键词： exuviae Genome editing identification locusts molt non-invasive

Engineered biocontainable RNA virus vectors for non-transgenic Genome editing across crop species and genotypes

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Molecular Plant 2023年第3期16卷 616-631页

作者： Qian Liu Chenglu Zhao Kai Sun Yinlu Deng Zhenghe Li State Key Laboratory of Rice Biology Institute of BiotechnologyZhejang UniversityHangzhouChina Ministry of Agricutture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests Zhejiang UniversityHangzhouChina Key Laboratory of Biology of Crop Pathogens and Insects of Zhejang Province Zhejang UniversityHangzhouChina

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science.Here,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant species.We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts.The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors.In systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency.Plants with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection.Moreover,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable mutations.Because many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for Genome editing in various crop species and elite varieties.

关键词： Genome editing CRISPR-Cas base editor RNA virus vector tomato spotted wilt virus bunyavirus delivery non-transgenic

Computational Tools and Resources for CRISPR/Cas Genome editing

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Genomics, Proteomics & Bioinformatics 2023年第1期21卷 108-126页

作者： Chao Li Wen Chu Rafaqat Ali Gill Shifei Sang Yuqin Shi Xuezhi Hu Yuting Yang Qamar U.Zaman Baohong Zhang Oil Crops Research Institute Chinese Academy of Agricultural SciencesKey Laboratory for Biology and Genetic Improvement of Oil CropsMinistry of Agriculture and Rural AffairsWuhan 430062China Graduate School of Chinese Academy of Agricultural Sciences Beijing 100081China Department of Biology East Carolina UniversityGreenvilleNC 27858USA

The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)nucleases and their functional variants,as well as in developing precise CRISPR/Cas-derived genome editors.The programmable and robust features of the genome editors provide an effective RNAguided platform for fundamental life science research and subsequent applications in diverse scenarios,including biomedical innovation and targeted crop improvement.One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts,which strongly depends on the efficiency and specificity of single guide RNA(sgRNA)-directed recognition of targeted DNA sequences.Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction.In this review,we first briefly introduce the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes.Secondly,we focus on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on-and off-target mutations.Thirdly,we provide insights into the limitations of available computational tools that would help researchers of this field for further optimization.Lastly,we suggest a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas Genome editing.

关键词： Genome editing Efficiency and specificity CRISPR/Cas9 sgRNA Computational tool Algorithm

Efficient CRISPR/Cas9-mediated Genome editing in sheepgrass(Leymus chinensis)

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Journal of Integrative Plant Biology 2023年第11期65卷 2416-2420页

作者： Zhelong Lin Lei Chen Shanjie Tang Mengjie Zhao Tong Li Jia You Changqing You Boshu Li Qinghua Zhao Dongmei Zhang Jianli Wang Zhongbao Shen Xianwei Song Shuaibin Zhang Xiaofeng Cao State Key Laboratory of Plant Genomics and National Center for Plant Gene Research Institute of Genetics and Developmental BiologyThe Chinese Academy of SciencesBeijing 100101China Institute of Forage and Grassland Sciences Heilongjiang Academy of Agricultural SciencesHarbin 150086China State Key Laboratory of Plant Cell and Chromosome Engineering Center for Genome EditingInstitute of Genetics and Developmental BiologyThe Chinese Academy of SciencesBeijing 100101China

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.

关键词： Agrobacterium genetic transformation Genome editing Lc TB1 sheepgrass

Efficient Genome editing in Claviceps purpurea using a CRISPR/Cas9 ribonucleoprotein method

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Synthetic and Systems Biotechnology 2022年第2期7卷 664-670页

作者： Lu Yu Meili Xiao Zhihua Zhu Yinmei Wang Zhihua Zhou Pingping Wang Gen Zou CAS-Key Laboratory of Synthetic Biology CAS Center for Excellence in Molecular Plant SciencesInstitute of Plant Physiology and EcologyChinese Academy of Sciences300 Fenglin RdShanghai200032China University of Chinese Academy of Sciences Beijing100049China Shanghai Key Laboratory of Agricultural Genetics and Breeding Institute of Edible FungiShanghai Academy of Agriculture Science1000 Jinqi RdFengxianShanghai201403China

Claviceps purpurea produces many pharmacologically important ergot alkaloids(EAS),which are widely used to treat migraine and hypertension and to aid childbirth.Although an EAS biosynthetic cluster of C.purpurea has been discovered more than 20 years ago,the complete biosynthetic pathway of EAS has not been fully characterized until now.The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C.purpurea.The conventional gene manipulation method for C.purpurea relies on homologous recombination(HR),although the efficiency of HR in C.purpurea is very low(~1-5%).Consequently,the disruption of target genes is laborious and time-consuming.Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently,their gene-disruption efficiency is still very low.Here,we developed an efficient genome-editing system in C.purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes.As proof of principle,three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system,with editing efficiencies ranging from 50%to 100%.Inactivation of the three genes,which are closely related to uridine biosynthesis(ura5),hypha morphology(rac),and EAS production(easA),resulted in a uridine auxotrophic mutant,a mutant with a drastically different phenotype in axenic culture,and a mutant that did not produce EAS,respectively.Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for Genome editing in C.purpurea,which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C.purpurea.

关键词： Ribonucleoprotein Genome editing Ergot alkaloids Biosynthetic pathway Homologous recombination

Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves Genome editing

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Science China(Life Sciences) 2022年第4期65卷 731-738页

作者： Fengti Qiu Sinian Xing Chenxiao Xue Jinxing Liu Kunling Chen Tuanyao Chai Caixia Gao State Key Laboratory of Plant Cell and Chromosome Engineering Center for Genome EditingInstitute of Genetics and Developmental BiologyInnovation Academy for Seed DesignChinese Academy of SciencesBeijing100101China College of Life Sciences University of Chinese Academy of SciencesBeijing100101China College of Advanced Agricultural Sciences University of Chinese Academy of SciencesBeijing100101China

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.

关键词： mTaGRF4-TaGIF1 wheat regeneration Genome editing transient expression

Recent advances in CRISPR-based Genome editing technology and its applications in cardiovascular research

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Military Medical Research 2023年第6期10卷 862-880页

作者： Zhen-Hua Li Jun Wang Jing-Ping Xu Jian Wang Xiao Yang State Key Laboratory of Proteomics Beijing Proteome Research CenterNational Center for Protein SciencesBeijing Institute of LifeomicsBeijing 100071China Yaneng BIOScience(Shenzhen)Co. Ltd.Shenzhen 518102GuangdongChina

The rapid development of Genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats(CRISPR)-based Genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein(Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided Genome editing systems have been uncovered, adding myriads of potential new tools to the Genome editing toolbox. CRISPR-based Genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel Genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise Genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based Genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases(CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of Genome editing technologies are discussed.

关键词： Genome editing CRISPR-Cas system Base editing Prime editing Transposon-associated Genome editing Cardiovascular disease Heart Blood vessel Gene therapy

Genome editing in cancer:Challenges and potential opportunities

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生物活性材料(英文) 2023年第3期21卷 394-402页

作者： Dor Breier Dan Peer Laboratory of Precision Nanomedicine Shmunis School of Biomedicine and Cancer ResearchGeorge S.Wise Faculty of Life SciencesTel Aviv UniversityTel AvivIsrael Department of Materials Sciences and Engineering Iby and Aladar Fleischman Faculty of EngineeringTel Aviv UniversityTel AvivIsrael Center for Nanoscience and Nanotechnology Tel Aviv UniversityTel AvivIsrael Cancer Biology Research Center Tel Aviv UniversityTel AvivIsrael

Ever since its mechanism was discovered back in 2012,the CRISPR/Cas9 system have revolutionized the field of Genome editing.While at first it was seen as a therapeutic tool mostly relevant for curing genetic diseases,it has been recently shown to also hold the potential to become a clinically relevant therapy for cancer.However,there are multiple challenges that must be addressed prior to clinical testing.Predominantly,the safety of the system when used for in-vivo therapies,including off-target activity and the effects of the double strand break induction on genomic stability.Here,we will focus on the inherent challenges in the CRISPR/Cas9 system and discuss various opportunities to overcoming these challenges.

关键词： Genome editing CRISPR/Cas systems Cancer therapy Off-target activity