Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters

Yanfei Liu,Zhenchang Liang,Peige Fan,Shaohua Li,Wei Duan,Yuchen Guo,Chong Ren

doi:10.1038/s41438-021-00489-z

Abstract

The efficacy of the CRISPR/Cas9 system in grapevine (Vitis vinifera L.) has been documented, but the optimization of this system, as well as CRISPR/Cas9-mediated multiplex genome editing, has not been explored in this species. Herein, we identified four VvU3 and VvU6 promoters and two ubiquitin (UBQ) promoters in grapevine and demonstrated that the use of the identified VvU3/U6 and UBQ2 promoters could significantly increase the editing efficiency in grape by improving the expression of sgRNA and Cas9, respectively. Furthermore, we conducted multiplex genome editing using the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette (PTG) by targeting the sugar-related tonoplastic monosaccharide transporter (TMT) family members TMT1 and TMT2, and the overall editing efficiencies were higher than 10%. The simultaneous editing of TMT1 and TMT2 resulted in reduced sugar levels, which indicated the role of these two genes in sugar accumulation in grapes. Moreover, the activities of the VvU3, VvU6, and UBQ2 promoters in tobacco genome editing were demonstrated by editing the phytoene desaturase (PDS) gene in Nicotiana benthamiana leaves. Our study provides materials for the optimization of the CRISPR/Cas9 system. To our knowledge, our simultaneous editing of the grape TMT family genes TMT1 and TMT2 constitutes the first example of multiplex genome editing in grape. The multiplex editing systems described in this manuscript expand the toolbox of grape genome editing, which would facilitate basic research and molecular breeding in grapevine.

Highlights

The advent of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been revolutionizing genome editing and genetic therapy[1,2]
The promoter regions of these genes were divergent between the two species, with the exception of the upstream sequence element (USE) and TATA-like box, which are required for transcription (Fig. 1a)
The expression of single guide RNA (sgRNA) and Cas[9] in transgenic grape cells was measured by quantitative real-time PCR (qRT-PCR), and the results showed that the transcript levels of the phytoene desaturase (PDS) sgRNA driven by the VvU3 and VvU6 promoters were higher (>3-fold) than those obtained using the AtU6 promoter (Fig. 4a)

Summary

Introduction

The advent of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been revolutionizing genome editing and genetic therapy[1,2]. CRISPR/Cas[9] has been more rapidly and widely applied in numerous plant species due to its simplicity, high efficiency, and versatility compared with previous genome editing technologies[3,4,5]. The most popular CRISPR/Cas[9] system was initially derived from the adaptive immune system in Streptococcus pyogenes[6], and an engineered system in which a single guide RNA (sgRNA) and the Cas[9] nuclease are needed for genome editing[1]. In the CRISPR/Cas[9] system, the expression of Cas[9] is generally driven by an RNA polymerase II (Pol II) promoter. During the early applications of the CRISPR/Cas[9] system in plants, the CaMV35S promoter was often used to express the Cas[9] gene[10,11,12]. The Pol III promoters of small nuclear RNA (snRNA) genes, such as

Methods

Results

Conclusion