SRP125727 PRJNA420019 gRNA validation for wheat genome editing with the CRISPR-Cas9 system 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. The objective of this study was to establish a rapid and reliable method for assessing gRNA activity and specificity in hexaploid wheat protoplasts. Seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were assessed. EPSPS is the biological target of the widely used herbicide glyphosate. Following transient co-expression of Cas9 and gRNA in protoplasts, targeted indels produced through NHEJ were detected via NGS.