High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize.

Weiwei Qi,Rentao Song,Chaobin Li,Zhongrui Tian,Wei Zhang,Tong Zhu

doi:10.1186/s12896-016-0289-2

Weiwei Qi, Rentao Song + Show 4 more

Open Access

https://doi.org/10.1186/s12896-016-0289-2

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Abstract

BackgroundCRISPR/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice.ResultsWe optimized and introduced the multiplex gene editing strategy based on the tRNA-processing system into maize. Maize glycine-tRNA was selected to design multiple tRNA-gRNA units for the simultaneous production of numerous gRNAs under the control of one maize U6 promoter. We designed three gRNAs for simplex editing and three multiple tRNA-gRNA units for multiplex editing. The results indicate that this system not only increased the number of targeted sites but also enhanced mutagenesis efficiency in maize. Additionally, we propose an advanced sequence selection of gRNA spacers for relatively more efficient and accurate chromosomal fragment deletion, which is important for complete abolishment of gene function especially long non-coding RNAs (lncRNAs). Our results also indicated that up to four tRNA-gRNA units in one expression cassette design can still work in maize.ConclusionsThe examples reported here demonstrate the utility of the tRNA-processing system-based strategy as an efficient multiplex genome editing tool to enhance maize genetic research and breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0289-2) contains supplementary material, which is available to authorized users.

Highlights

clustered regularly interspersed short palindromic repeat (CRISPR)/Cas9 genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple guide RNA (gRNA) into one synthetic gene for manipulating multiple genes in rice
We found the tRNA-processing system-based method improves the efficiency of CRISPR/Cas9 editing in maize and proposed an advanced gRNA selection strategy for chromosomal fragment deletion purpose
Strategy to engineer simplex editing and multiplex editing based on the tRNA-processing system in maize A maize codon optimized Cas9 driven by the maize ubiquitin (UBQ) promoter was inserted into pCAMBIA3301 to construct two binary CRISPR/Cas9 vectors for either simplex editing or multiplex editing (Fig. 1)

Summary

Introduction

CRISPR/Cas genome editing strategy has been applied to a variety of species and the tRNA-processing system has been used to compact multiple gRNAs into one synthetic gene for manipulating multiple genes in rice. The Cas gene and a 20-bp guide RNA (gRNA) that is complementary to the DNA site being targeted for mutation need to be transformed into the target organism to create a gene disruption. Gene editing tools with the capability to manipulate multiple targets are of great value. Multiplex gene editing can be achieved by expressing Cas along with multiple gRNAs, each targeting different sites. Conventional delivery methods involve creating gene constructs containing multiple gRNA expressing cassettes for multiplex gene editing in one plasmid or using multiple plasmids [15,16,17,18,19,20]. Due to the limitations of the delivery method and plasmid capacity, compacting multiple gRNAs into one synthetic gene would be an

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