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multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces

Science China(Life Sciences) 2020年第7期63卷 1053-1062页

作者： Yawei Zhao Jinzhong Tian Guosong Zheng Jun Chen Chuanwen Sun Zhongyi Yang Andrei A.Zimin Weihong Jiang Zixin Deng Zhijun Wang Yinhua Lu College of Life Sciences.Shanghai Normal University Shanghai 200234China State Key Laboratory of Microbial Metabolism School of Life Sciences and Biotechnology Shanghai Jiao Tong UniversityShanghai 200030China Key Laboratory of Synthetic Biology CAS Center for Excellence in Molecular Plant SciencesShanghai Institute of Plant Physiology and EcologyChinese Academy of SciencesShanghai 200032China School of Life Science Taizhou UniversityTaizhou 318000China Institute of Biochemistry and Physiology of Microorganisms Russian Academy of SciencesPushchino 142290Russia Shanghai Engineering Research Center of Plant Germplasm Resources College of Life SciencesShanghai Normal UniversityShanghai 200234China

CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination(HR) ability. Here, a deaminase-assisted base editor d Cas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9(dCas9), the cytidine deaminase from Petromyzon marinus(PmCDA1), the uracil DNA glycosylase inhibitor(UGI) and the protein degradation tag(LVA tag). Using d Cas9-CDA-ULstr , we achieved single-, double-and triple-point mutations(cytosine-to-thymine substitutions)at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes(with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the d Cas9-CDA-ULstr -based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.

关键词： base editors dCas9-CDA-ULstr multiplex editing Streptomyces

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DRISPR/Cas9 Platforms for Genome editing in Plants： Developments and Applications

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Molecular Plant 2016年第7期9卷 961-974页

作者： Xingliang Ma Qinlong Zhu Yuanling Ghen Yao-Guang Liu State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources Guangzhou 510642 China Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions Guangzhou 510642 China College of Life Sciences South China Agricultural University Guangzhou 510642 China

The clustered regularly interspaced short palindromic repeat （CRISPR）-associated protein9 （Cas9） genome editing system （CRISPR/Casg） is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adap- tion of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/ Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, fac- tors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.

关键词： CRISPR/Cas9 genome editing multiplex editing mutation plants

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Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture.

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Molecular Plant 2021年第11期14卷 1787-1798页

作者： Tingdong Li Jiacheng Hu Yu Sun Boshu Li Dingliang Zhang Wenli Li Jinxing Liu Dawei Li Caixia Gao Yongliang Zhang Yanpeng Wang State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology College of Biological SciencesChina Agricultural UniversityBeijingChina State Key Laboratory of Plant Cell and Chromosome Engineering Center for Genome EditingInstitute of Genetics and Developmental BiologyThe Innovative Academy of Seed DesignChinese Academy of SciencesBeijingChina College of Advanced Agricultural Sciences University of Chinese Academy of SciencesBeijingChina College of Life Sciences University of Chinese Academy of SciencesBeijingChina

Genome editing provides novel strategies for improving plant traits,but relies on current genetic transformation and plant regeneration procedures,which can be *** 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 *** 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 *** also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different ***,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 *** study provides a robust,convenient and tissue culture-free approach for genome editing in wheat through virus infection.

关键词： heritable genome editing BSMV common wheat tissue culture-free multiplex editing

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Efficient genome editing in grapevine using CRISPR/LbCas12a system

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Molecular Horticulture 2023年第1期3卷 111-123页

作者： Chong Ren Elias Kirabi Gathunga Xue Li Huayang Li Junhua Kong Zhanwu Dai Zhenchang Liang Beijing Key Laboratory of Grape Sciences and Enology Beijing 100093PR China State Key Laboratory of Plant Diversity and Specialty Crops Beijing 100093PR China China National Botanical Garden Beijing 100093PR China University of Chinese Academy of Sciences Beijing 100049PR China Institute of Botany the Chinese Academy of SciencesHaidian DistrictNanxin Village 20XiangshanBeijing 100093China

Clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system,also known as CRISPR/Cpf1,has been successfully harnessed for genome engineering in many plants,but not in grapevine *** we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006(LbCas12a)in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1(TMT1)and dihydroflavonol-4-reductase 1(DFR1)genes in 41B *** of DFR1 gene altered flavonoid accumulation in dfr1 mutant *** treatment(34℃)improved the editing efficiencies of CRISPR/LbCas12a system,and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3%to 44.6%and 29.9%to 37.3%after heat treatment,***,the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome *** addition,genome editing with truncated crRNAs(trucrRNAs)showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis,whereas trucrRNAs with shorter regions of target complementarity≤18 nt in length may not induce detectable mutations in 41B *** these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.

关键词： Vitis vinifera CRISPR LbCas12a multiplex editing crRNA length Flavonoid

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Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

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aBIOTECH 2020年第2期1卷 123-134页

作者： Vicki Hsieh-Feng Yinong Yang Intercollege Graduate Degree Program in Plant Biology The Huck Institutes of the Life SciencesThe Pennsylvania State UniversityUniversity ParkPA 16802USA Department of Plant Pathology and Environmental Microbiology The Pennsylvania State UniversityUniversity ParkPA 16802USA

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various *** efficient and proper expression of multiple guide RNAs(gRNAs),the CRISPR/Cas system is particularly suitable for multiplex genome *** the past several years,different CRISPR/Cas expression strategies,such as two-component transcriptional unit,single transcriptional unit,and bidirectional promoter systems,have been developed to efficiently express gRNAs as well as Cas *** progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes,endogenous RNases,and exogenous endoribonuclease(Csy4).Besides being constitutively and ubiquitously expressed,inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible,tissue-specific,and/or synthetic promoters for specific research *** recently,the emergence of CRISPR/Cas ribonucleoprotein delivery methods,such as engineered nanoparticles,further revolutionized transgene-free and multiplex genome *** this review,we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.

关键词： CRISPR/Cas Gene expression Genome engineering Guide RNA multiplex editing

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