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gRNA validation for wheat genome editing with the CRISPR-Cas9 system.

Abstract：

BACKGROUND:The 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. RESULTS:The 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. CONCLUSIONS:We 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.

journal_name

BMC Biotechnol

journal_title

BMC biotechnology

authors

Arndell T,Sharma N,Langridge P,Baumann U,Watson-Haigh NS,Whitford R

doi

10.1186/s12896-019-0565-z

subject

Has Abstract

pub_date

2019-10-30 00:00:00

pages

71

issue

1

issn

1472-6750

pii

10.1186/s12896-019-0565-z

journal_volume

19

pub_type

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