Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA
| Autor: | Masao Ishimoto, Yuhei Kanazashi, Jun Abe, Tetsuya Yamada, Sakiko Hirose, Seiichi Toki, Masaki Endo, Akito Kaga, Aya Hirose, Ippei Takahashi, Ken Naito, Masafumi Mikami |
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| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Mutant Inheritance Patterns Mutagenesis (molecular biology technique) Plant Science Biology Genes Plant medicine.disease_cause 01 natural sciences Frameshift mutation 03 medical and health sciences Genes Duplicate medicine Arabidopsis thaliana CRISPR Amino Acid Sequence Site-directed mutagenesis Plant Proteins Genetics Mutation Sequence Homology Amino Acid Cas9 fungi food and beverages General Medicine Plants Genetically Modified biology.organism_classification DNA-Binding Proteins 030104 developmental biology Mutagenesis Site-Directed Soybeans CRISPR-Cas Systems Agronomy and Crop Science Genome Plant RNA Guide Kinetoplastida 010606 plant biology & botany |
| Zdroj: | Plant Cell Reports. 37:553-563 |
| ISSN: | 1432-203X 0721-7714 |
| DOI: | 10.1007/s00299-018-2251-3 |
| Popis: | Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T1 and T2 generations indicates that putative mutation induced in the T0 plant is transmitted to the T1 generation. The inheritable mutation induced in the T1 plant was also detected. This result indicates that continuous induction of mutations during T1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean. |
| Databáze: | OpenAIRE |
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