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A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants

The Crop Journal 2024年第2期12卷 569-582页

作者： Suting Wu Htin Kyaw Zhijun Tong Yirong Yang Zhiwei Wang Liying Zhang Lihua Deng Zhiguo Zhang Bingguang Xiao William Paul Quick Tiegang Lu Guoying Xiao Guannan Qin Xue'an Cui Biotechnology Research Institute Chinese Academy of Agricultural SciencesBeijing 100081China Key Laboratory of Genetics Breeding and Multiple Utilization of CropsMinistry of EducationCollege of AgricultureFujian Agriculture and Forestry UniversityFuzhou 350002FujianChina Key Laboratory of Biological Breeding for Fujian and Taiwan Crops Ministry of Agriculture and Rural AffairsCollege of AgricultureFujian Agriculture and Forestry UniversityFuzhou 350002FujianChina Key Laboratory of Agro-ecological Processes in Subtropical Region Institute of Subtropical AgricultureChinese Academy of SciencesChangsha 410125HunanChina School of Biosciences The University of SheffieldFirth CourtWestern BankSheffield S102TNUK Key Laboratory of Tobacco Biotechnological Breeding National Tobacco Genetic Engineering Research CenterYunnan Academy of Tobacco Agricultural SciencesKunming 650021YunnanChina

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic *** available genome-editing tools have a limited number of targets,restricting their application in genetic *** this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software *** combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was *** plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

关键词： CRISPR/Cas9 multiplex genome editing Assembly system Plant

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Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

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Plant Communications 2024年第2期5卷 67-78页

作者： Ajay Gupta Bo Liu Saad Raza Qi-Jun Chen Bing Yang Division of Plant Science and Technology Bond Life Sciences CenterUniversity of MissouriColumbiaMO 65211USA State Key Laboratory of Plant Physiology and Biochemistry College of Biological SciencesChina Agricultural UniversityBeijing 100193China Center for Crop Functional Genomics and Molecular Breeding China Agricultural UniversityBeijing 100193China Donald Danforth Plant Science Center St.LouisMO 63132USA

Prime editing(PE)technology enables precise alterations in the genetic code of a genome of *** offers great potential for identifying major agronomically important genes in plants and editing them into superior variants,ideally targeting multiple loci simultaneously to realize the collective effects of the ***,we report the development of a modular assembly-based multiplex PE system in rice and demon-strate its efficacy in editing up to four genes in a single transformation *** duplex PE(DPE)system achieved a co-editing efficiency of 46.1%in the T0 generation,converting TFIIAg5 to xa5 and xa23 to *** resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae(Xoo)strains in the T1 *** addition,we success-fully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele,achieving a co-editing rate of 57.14%.Furthermore,with the quadruple PE(QPE)system,we edited four genes—two for herbicide tolerance(OsEPSPS1 and OsALS1)and two for Xoo resistance(TFIIAg5 and OsSWEET11a)—using one construct,with a co-editing efficiency of 43.5%for all four genes in the T0 *** performed multiplex PE usingfive more constructs,including two for triplex PE(TPE)and three for QPE,each targeting a different set of *** editing rates were dependent on the activity of pegRNA and/or *** instance,optimization of ngRNA increased the PE rates for one of the targets(OsSPL13)from 0%to 30%but did not improve editing at another target(OsGS2).Overall,our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents,making it feasible for more labs to utilize PE for their editing *** have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.

关键词： prime editing rice bacterial blight herbicide tolerance multiplex genome editing

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