Bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome Editing

Qiurong Ren; Zhaohui Zhong; Yan Wang; Qi You; Qian Li; Mingzhu Yuan; Yao He; Caiyan Qi; Xu Tang; Xuelian Zheng; Tao Zhang; Yiping Qi; Yong Zhang

doi:10.3389/fpls.2019.01173

Bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome Editing

Front Plant Sci. 2019 Sep 20:10:1173. doi: 10.3389/fpls.2019.01173. eCollection 2019.

Authors

Qiurong Ren¹, Zhaohui Zhong¹, Yan Wang¹, Qi You^{2

3}, Qian Li¹, Mingzhu Yuan¹, Yao He¹, Caiyan Qi¹, Xu Tang¹, Xuelian Zheng¹, Tao Zhang^{2

3}, Yiping Qi^{4

5}, Yong Zhang^{1

2}

Affiliations

¹ Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China.
² Jiangsu Key Laboratory of Crop Genetics and Physiology, Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou, China.
³ Key Laboratory of Plant Functional Genomics of the Ministry of Education, Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou University, Yangzhou, China.
⁴ Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, United States.
⁵ Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, United States.

Abstract

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.

Keywords: CRISPR-Cas9; bidirectional promoter; enhancer; plant genome editing; rice.