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

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Agricultural biotechnology

Background:

• CRISPR-Cas9 is a key tool for crop genome editing, but efficiency and specificity are challenging in polyploid species like wheat.
• Guide RNA (gRNA) performance is critical for CRISPR-Cas9 system efficacy.
• The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene is the target of the herbicide glyphosate.

Purpose of the Study:

• To assess the activity and specificity of seven gRNAs targeting the EPSPS gene in hexaploid wheat.
• To evaluate the correlation between in silico predictions and experimental results for gRNA efficiency.
• To identify suitable gRNAs for developing non-transgenic glyphosate-resistant wheat.

Main Methods:

• Tested seven gRNAs targeting the EPSPS gene in hexaploid wheat protoplasts.
• Quantified on-target editing efficiency by measuring indel frequencies.
• Analyzed the impact of mismatches and PAM sequence on gRNA activity.

Main Results:

• Significant variation in on-target activity among the seven gRNAs, with indel frequencies from 0% to ~20%.
• No clear correlation observed between predicted and experimentally determined gRNA on-target activity.
• Single mismatches reduced activity, while additional mismatches or absent PAM nearly abolished it; large insertions were detected.

Conclusions:

• Developed a rapid, reliable method for gRNA validation in wheat protoplasts, suitable for polyploids and other species.
• Identified a specific gRNA with properties suitable for developing non-transgenic glyphosate-resistant wheat.
• Experimental validation of gRNAs is crucial for optimizing CRISPR-Cas9 applications in plants.