A Simple and Low-Tech Heat-Shock Method to Increase Genome Editing Efficiency in Plants

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas is a standard tool for creating new plant genotypes.
• Optimizing CRISPR efficiency is crucial for its broader application.
• Heat stress has been shown to increase mutagenesis efficiency in plants.

Purpose of the Study:

• To develop a simplified, low-tech heat stress assay for in vitro plant systems.
• To increase the efficiency of CRISPR/Cas-induced mutagenesis in plants.
• To provide accessible methods for quantifying genome editing efficiency.

Main Methods:

• A simplified heat stress assay using three 24-hour heat shocks at 37°C with 24-hour recovery periods at 21°C.
• Utilizing visual mutant phenotypes (pds3 and gl1) for assessing genome editing.
• Quantifying genome editing efficiency via genotyping by Sanger sequencing.
• A one-step cloning protocol for CRISPR expression vectors.

Main Results:

• The 3x heat shock protocol significantly increased indel rates for LbCas12a and Cas9.
• Visual phenotypes and Sanger sequencing effectively quantified genome editing efficiency.
• The entire heat stress assay can be completed within 6 days.
• A streamlined cloning method was successfully developed.

Conclusions:

• The described low-tech heat stress method efficiently enhances CRISPR-induced mutations in vitro.
• This protocol simplifies heat stress application and phenotyping for plant genome editing.
• The methods presented facilitate increased mutation rates and efficient quantification in plant research.