Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

Yi Zhang,Yanpeng Wang,Zhen Liang,Jinxing Liu,Kunling Chen,Yuan Zong,Caixia Gao,Jin-Long Qiu

doi:10.1038/ncomms12617

Abstract

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

Highlights

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns
We demonstrate a simple and efficient genome-editing approach by which mutant plants are regenerated after transiently expressing CRISPR/Cas[9] DNA, or through transient expression of the in vitro transcripts (IVTs) of Cas9-coding sequence and guide RNA (TECCRNA hereafter), into wheat callus cells
We found that these genome-editing methods are highly efficient for both hexaploid bread wheat (Triticum aestivum L., AABBDD, 2n 1⁄4 6x 1⁄4 42) and tetraploid durum wheat (T. turgidum L. var. durum, AABB, 2n 1⁄4 4x 1⁄4 28), and should be applicable to other plant species

Summary

Introduction

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. We report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas[9] introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. Our tissue culture procedures are free of lengthy, costly and labour-intensive herbicide selection (Fig. 1), and homozygous mutant plants with no detectable transgenes are identified in T0 populations We found that these genome-editing methods are highly efficient for both hexaploid bread wheat (Triticum aestivum L., AABBDD, 2n 1⁄4 6x 1⁄4 42) and tetraploid durum wheat We found that these genome-editing methods are highly efficient for both hexaploid bread wheat (Triticum aestivum L., AABBDD, 2n 1⁄4 6x 1⁄4 42) and tetraploid durum wheat (T. turgidum L. var. durum, AABB, 2n 1⁄4 4x 1⁄4 28), and should be applicable to other plant species

Methods

Results

Conclusion