An Improved CRISPR/Cas9 System for Genome Editing in Populus by Using Mannopine Synthase (MAS) Promoter.

Yi An,Juan Du,Chun Wang,Junguang Yao,Ya Geng

doi:10.3389/fpls.2021.703546

Abstract

Gene editing technology in woody plants has great potential for understanding gene function, and altering traits affecting economically and ecologically important traits. Gene editing applications in woody species require a high genome editing efficiency due to the difficulty during transformation and complexities resulting from gene redundancy. In this study, we used poplar 84K (Populus alba × P. glandulosa), which is a model hybrid for studying wood formation and growth. We developed a new CRISPR/Cas9 system to edit multiple genes simultaneously. Using this system, we successfully knocked out multiple targets of the PHYTOENE DESATURASE 8 in poplar. We found the mutation rate of our CRISPR/Cas9 system is higher (67.5%) than existing reports in woody trees. We further improved the mutation rate up to 75% at editing sites through the usage of the mannopine synthase (MAS) promoter to drive Cas9. The MAS-CRISPR/Cas9 is an improved genome-editing tool for woody plants with a higher efficiency and a higher mutation rate than currently available technologies.

Highlights

Poplars (Populus) have tremendous economic and ecological value because of their timber, bioenergy applications, rapid pulp production rotation, as well as their key ecological roles in temperate forests across the northern hemisphere (Jansson and Douglas, 2007; Polle et al, 2013)
Cassettes were driven by the Arabidopsis RNA polymerase III promoter AtU6-26 while Cas9 was driven by the 2 × 35S promoter or mannopine synthase (MAS) promoter in pCambia1300 binary vectors (Figure 2A)
The three gRNAs were inserted into the pC1300-2 × 35S-Cas9 and pC1300-MAS-Cas9 binary vectors between the Kpn I and BamH I sites by a one-step ligation (Figure 2B)

Summary

Introduction

Poplars (Populus) have tremendous economic and ecological value because of their timber, bioenergy applications, rapid pulp production rotation, as well as their key ecological roles in temperate forests across the northern hemisphere (Jansson and Douglas, 2007; Polle et al, 2013). Three kinds of Populus have been edited via CRISPR/Cas, including Populus tomentosa Carr (Fan et al, 2015), 717 (Populus tremula × P. alba) (Zhou et al, 2015), and Shanxin yang (Populus davidiana × P. bolleana) (Wang et al, 2020). Cultivated in China and Korea, the hybrid clone 84K (Populus alba × P. glandulosa) is a model hybrid for the study wood formation and stress response and has relatively high rates of transformation compared with other woody plants (Li et al, 2017; Qiu et al, 2019). The recent availability of poplar 84K genome sequence facilitates functional genomic studies and the ability to design

Methods

Results

Conclusion