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Bidirectional promoter based CRISPR-Cas9 systems for plant genome editing
Friday, 2019/09/06 | 08:33:34

Qiurong RenZhaohui Zhong, Yan Wang, Qi You, Qian Li, Mingzhu Yuan, Yao He, Caiyan Qi, Xu TangXuelian ZhengTao ZhangYiping Qi and Yong Zhang

Front. Plant Sci. | doi: 10.3389/fpls.2019.01173; Received: 20 Feb 2019; Accepted: 27 Aug 2019.




Figure: A bidirectional promoter system (BiP) was developed for genome editing, which allows expression of Cas9 and single guide RNA (sgRNA) in opposite directions. This was developed by experts from the University of Electronic Science and Technology of China, Yangzhou University, and the University of Maryland.


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 promoter future research and adoption of BiP systems for CRISPR-Cas based genome engineering in plants.


See https://www.frontiersin.org/articles/10.3389/fpls.2019.01173/abstract

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