A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants
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
Affiliations
Suting Wu
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Htin Kyaw
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Zhijun Tong
Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650021, Yunnan, China
Yirong Yang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Zhiwei Wang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Liying Zhang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Lihua Deng
Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, Hunan, China
Zhiguo Zhang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Bingguang Xiao
Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Yunnan Academy of Tobacco Agricultural Sciences, Kunming 650021, Yunnan, China
William Paul Quick
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; School of Biosciences, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK
Tiegang Lu
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Guoying Xiao
Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, Hunan, China; Corresponding authors.
Guannan Qin
Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education, College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; Key Laboratory of Biological Breeding for Fujian and Taiwan Crops, Ministry of Agriculture and Rural Affairs, College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China; Corresponding authors.
Xue’an Cui
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; Corresponding authors.
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.
