A streamlined guide RNA screening system for genome editing in Sorghum bicolor

Area of Science:

• Plant biotechnology
• Molecular biology
• Genomics

Background:

• CRISPR-Cas systems offer powerful tools for targeted plant genome editing.
• Variable efficiency of single guide RNAs (sgRNAs) hinders rapid crop improvement.
• A need exists for efficient methods to evaluate sgRNA activity in plants.

Purpose of the Study:

• To develop and optimize a rapid screening system for evaluating sgRNA efficiency in sorghum genome editing.
• To compare different CRISPR delivery methods (plasmid vs. RNP) in sorghum protoplasts.
• To establish optimal conditions for sorghum protoplast isolation and transfection.

Main Methods:

• Developed a transient protoplast transfection assay for sorghum.
• Optimized sorghum plant cultivation for high-yield protoplast isolation.
• Utilized polyethylene glycol-mediated transfection for delivering CRISPR components.
• Analyzed indel frequencies using targeted deep sequencing.

Main Results:

• Optimized cultivation conditions yielded high numbers of viable sorghum protoplasts with efficient transfection.
• Plasmid-mediated delivery achieved up to 77.8% indel frequency, while RNP delivery reached 18.5%.
• The entire screening process was completed within 16 days.

Conclusions:

• The developed sgRNA screening system is efficient and straightforward for sorghum genome editing.
• This system significantly reduces the time and resources required for sorghum crop improvement.
• Predicting sgRNA activity early accelerates the development of gene-edited sorghum varieties.