CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat |
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| Affiliation: | 1. Peking University Institute of Advanced Agricultural Sciences, Weifang, Shandong, 261325, China;2. State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Advanced Agriculture Sciences and School of Life Sciences, Peking University, Beijing, 100871, China;3. College of Life Sciences, Capital Normal University, Beijing Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement, Beijing, 100048, China;1. College of Life Science, Northeast Agricultural University, Harbin 150030, China;2. Northeast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding, Chinese Academy of Sciences, Harbin 150081, China;3. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;4. Rice Research Institute, College of Agriculture, Northeast Agricultural University, Harbin 150030, China;1. State Key Laboratory of Hybrid Rice, Shanghai Jiao Tong University–University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 20040, China;2. Plant Genomics Center, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, South Australia 5064, Australia;3. Department of Biology, East Carolina University, Greenville, NC 27858, USA;1. State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China;2. The Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China;3. State Key Laboratory of Plant Cell and Chromosome Engineering, and Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China;4. These authors contributed equally to this work;1. State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China, Sichuan Agricultural University, Chengdu, Sichuan 611130, China;2. Triticeae Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China;3. John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK;4. CSIRO Agriculture and Food, Black Mountain, Canberra, ACT 2601, Australia |
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| Abstract: | Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops. High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat. The emerging effective genome editing tool, CRISPR/Cas9 system, makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles. To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat, we compared four different RNA polymerase (Pol) III promoters (TaU3p, TaU6p, OsU3p, and OsU6p) and three types of sgRNA scaffold in the protoplast system. We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective. The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles, whose orthologs, OsNP1 in rice and ZmIPE1 in maize, encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility. Triple homozygous mutations in TaNP1 genes result in complete male sterility. We further demonstrated that any one wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility. Taken together, this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system. |
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| Keywords: | Wheat CRISPR/Cas9 Male sterility |
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