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

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement. Currently available genome-editing tools have a limited number of targets, restricting their application in genetic research. In this study, we devel...

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Bibliographic Details
Published in:The Crop journal 2024-04, Vol.12 (2), p.569-582
Main Authors: Wu, Suting, Kyaw, Htin, Tong, Zhijun, Yang, Yirong, Wang, Zhiwei, Zhang, Liying, Deng, Lihua, Zhang, Zhiguo, Xiao, Bingguang, Quick, William Paul, Lu, Tiegang, Xiao, Guoying, Qin, Guannan, Cui, Xue’an
Format: Article
Language:English
Subjects:
Assembly system
Cassettes
Cloning
Cloning vectors
computer software
CRISPR
CRISPR-Cas systems
CRISPR/Cas9
Design
DNA repair
Editing
Efficiency
Expression vectors
Fragments
gene editing
Gene expression
Genes
Genetic engineering
genetic improvement
genome
Genomes
Genomics
Metabolism
Multiplex genome editing
Multiplexing
Mutagenesis
Mutation
Plant
Plasmids
Proteins
Rice
synthetic biology
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Summary:The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement. Currently available genome-editing tools have a limited number of targets, restricting their application in genetic research. In 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 package. By 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 sgRNA 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 sgRNA expression cassettes was assembled and a high co-editing efficiency was observed. This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.
ISSN:2214-5141
2095-5421
2214-5141
DOI:10.1016/j.cj.2024.01.010