Journal
FRONTIERS IN PLANT SCIENCE
Volume 10, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fpls.2019.01173
Keywords
CRISPR-Cas9; plant genome editing; rice; bidirectional promoter; enhancer
Categories
Funding
- Sichuan Youth Science and Technology Foundation [2017JQ0005]
- National Science Foundation of China [81872957, 31771486]
- Program for International Science and Technology Cooperation and Exchanges of Sichuan Province [2018HH0112]
- Science Strength Promotion Program of UESTC
- National Transgenic Major Project [2018ZX08020-003]
- Open Foundation of Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding [PL201801]
- Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)
- National Science Foundation Plant Genome Research Program [IOS1758745]
- USDA-NIFA Biotechnology Risk Assessment Research Program [2018-33522-28789]
- Foundation for Food and Agriculture Research [593603]
- Syngenta Biotechnology Research Grant
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CRISPR-Cas systems can be expressed in multiple ways, with different capabilities regarding tissue-specific expression, efficiency, and expression levels. Thus far, three expression strategies have been demonstrated in plants: mixed dual promoter systems, dual Pol II promoter systems, and single transcript unit (STU) systems. We explored a fourth strategy to express CRISPR-Cas9 in the model and crop plant, rice, where a bidirectional promoter (BiP) is used to express Cas9 and single guide RNA (sgRNA) in opposite directions. We first tested an engineered BiP system based on double-mini 35S promoter and an Arabidopsis enhancer, which resulted in 20.7% and 52.9% genome editing efficiencies at two target sites in T0 stable transgenic rice plants. We further improved the BiP system drastically by using a rice endogenous BiP, OsBiP1. The endogenous BiP expression system had higher expression strength and led to 75.9-93.3% genome editing efficiencies in rice T0 generation, when the sgRNAs were processed by either tRNA or Csy4. We provided a proof-of-concept study of applying BiP systems for expressing two-component CRISPR-Cas9 genome editing reagents in rice. Our work could promote future research and adoption of BiP systems for CRISPR-Cas-based genome engineering in plants.
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