GRNA validation for wheat genome editing with the CRISPR-Cas9 system

Taj Arndell,Niharika Sharma,Ryan Whitford,Peter Langridge,Ute Baumann,Nathan S Watson-Haigh

doi:10.1186/s12896-019-0565-z

Abstract

BackgroundThe CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.ResultsThe seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.ConclusionsWe have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

Highlights

The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system is a powerful and versatile tool for crop genome editing
Homoeologous Single nucleotide polymorphism (SNP) located at the 3′ end of exon 2, in close proximity to a universal mutation hotspot for resistance to glyphosate [46] (Fig. 1)
In summary, this study demonstrates that guide RNA (gRNA) validation is an essential step in the application of the CRISPR-Cas9 system in wheat. gRNA validation should be carried out prior to commencing transformation and tissue culture experiments for the production of stably edited wheat plants

Summary

Introduction

The CRISPR-Cas system is a powerful and versatile tool for crop genome editing. achieving highly efficient and specific editing in polyploid species can be a challenge. Over the last few years, the field of genome editing has been revolutionised by the introduction of the CRISPR (clustered regularly-interspaced short palindromic repeats)-Cas (CRISPR associated protein) system [2,3,4,5,6,7]. This system consists of the Cas endonuclease in complex with a small guide RNA (gRNA) that is engineered to target a specific site in the genome. Flexibility, and high specificity, the CRISPR-Cas system has been and continues to be rapidly adopted by the plant research community for basic research and crop improvement

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