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

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

doi:10.3389/fpls.2019.01173

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.

Highlights

IntroductionCRISPR-Cas and Cas12a (formerly Cpf1) are widely used sequence-specific nucleases (SSNs) for plant genome editing (Jiang et al, 2013; Li et al, 2013; Nekrasov et al, 2013; Fauser et al, 2014; Endo et al, 2016; Xu et al, 2016; Begemann et al, 2017; Tang et al, 2017; Zhou et al, 2017; Lowder et al, 2018; Tang et al, 2018; Zhong et al, 2018; Zhong et al, 2019; Zhou et al, 2019)
To demonstrate that we can expression CRISPR-Cas9 with a bidirectional promoter (BiP), we first engineered a BiP based on CaMV 35s minimal promoter (Xie et al, 2001)
The results show that the mini 35s-enhancer-Csy4 BiP system resulted in detectable mutations, which were absent from the mini 35s-Csy4 negative control samples (Figures 1B, C)

Summary

Introduction

CRISPR-Cas and Cas12a (formerly Cpf1) are widely used sequence-specific nucleases (SSNs) for plant genome editing (Jiang et al, 2013; Li et al, 2013; Nekrasov et al, 2013; Fauser et al, 2014; Endo et al, 2016; Xu et al, 2016; Begemann et al, 2017; Tang et al, 2017; Zhou et al, 2017; Lowder et al, 2018; Tang et al, 2018; Zhong et al, 2018; Zhong et al, 2019; Zhou et al, 2019). Zinc finger nucleases (ZFNs), and TAL effector nucleases (TALENs), a CRISPRCas system relies on a single guide RNA (sgRNA, for Cas9) or CRISPR RNA (crRNA, for Cas12a) for DNA targeting, bypassing protein engineering. This easiness has made CRISPR-Cas systems as Bidirectional Promoter (BiP) CRISPR-Cas Systems top SSN choices for plant reverse genetics and accelerated crop breeding. Pol III promoters, such as U6 or U3, cannot match the expression strength of some strong Pol II promoters, which limits the overall genome editing efficiency in plants (Tang et al, 2016; Cermak et al, 2017; Mikami et al, 2017; Tang et al, 2019). Pol III promoters are only suitable for expression of relatively short transcripts, which prevents the use of a single Pol III promoter to effectively express multiple sgRNAs for multiplexed genome editing, an important feature and advantage of CRISPR-Cas technologies

Methods

Results

Conclusion