Genome Engineering in Plant Using an Efficient CRISPR-xCas9 Toolset With an Expanded PAM Compatibility.

Chengwei Zhang,Si Zhao,Yongxing Yang,Lu Li,Feipeng Wang,Guiting Kang,Jinxiao Yang,Xiang Zhang,Xinxiang Liu,Shuang Yuan

doi:10.3389/fgeed.2020.618385

Abstract

The CRISPR-Cas9 system enables simple, rapid, and effective genome editing in many species. Nevertheless, the requirement of an NGG protospacer adjacent motif (PAM) for the widely used canonical Streptococcus pyogenes Cas9 (SpCas9) limits the potential target sites. The xCas9, an engineered SpCas9 variant, was developed to broaden the PAM compatibility to NG, GAA, and GAT PAMs in human cells. However, no knockout rice plants were generated for GAA PAM sites, and only one edited target with a GAT PAM was reported. In this study, we used tRNA and enhanced sgRNA (esgRNA) to develop an efficient CRISPR-xCas9 genome editing system able to mutate genes at NG, GAA, GAT, and even GAG PAM sites in rice. We also developed the corresponding xCas9-based cytosine base editor (CBE) that can edit the NG and GA PAM sites. These new editing tools will be useful for future rice research or breeding, and may also be applicable for other related plant species.

Highlights

We revealed that the tRNA-enhanced sgRNA (esgRNA) system might help the xCas9-based cytosine base editor (CBE) to efficiently edit target sites with a GA protospacer adjacent motif (PAM) in rice (Zhang et al, 2020)
The rice codon-optimized xCas9 sequence with A262T/R324L/S409I/E480K/E543D/M694I/E1219V mutations (Zhang et al, 2020) under the control of the Oryza sativa ubiquitin (OsUbq) promoter was used in this study (Figure 1A)
We determined that tRNA increased the editing efficiency of xCas9 (Table 1 and Supplementary Table 4), which was consistent with the results of earlier studies in which the tRNA-sgRNA system enhanced the activity of high-fidelity CRISPR-associated nuclease 9 (Cas9) variants in human cells and rice (Zhang et al, 2017; He et al, 2019)

Summary

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system derived from microbial adaptive immune systems has facilitated diverse genomic manipulations, including targeted gene disruption (Bortesi and Fischer, 2015; Ma et al, 2015; Xie et al, 2015), transcriptional activation or repression (Lowder et al, 2015; Piatek et al, 2015), and base substitutions (Li et al, 2017; Lu and Zhu, 2017; Zong et al, 2017), in various organisms and cell types (Komor et al, 2017; Ge et al, 2019) The application of these genomic modifications has led to substantial advances in research regarding plant biology as well as crop breeding (Yin et al, 2017; Hille et al, 2018). LbCpf and FnCpf can modify the genomes of many plant species (Endo et al, 2016; Tang et al, 2017)

Methods

Results

Discussion

Conclusion