Crop Biotech Update

Simplified Heat Stress Assay Increases Mutagenesis Efficiency in CRISPR

December 7, 2022

Scientists from Ghent University were able to increase the mutagenesis efficiency of the gene editing tool CRISPR by alternating heat shocks and recovery. The method was found to be useful for Cas9 and LbCas12a systems.

CRISPR is a popular gene editing tool known for its precision and low-cost. Scientists continually try to improve the tool systems to achieve optimal results. Applying heat stress is one of the ways to increase CRISPR mutagenesis efficiency, but this usually requires a dedicated climate chamber that not all researchers have. To address this, a simple heat assay method was developed using commonly available laboratory equipment. The method takes six days to complete and involves three 24-hour heat shocks at 37 degrees Celsius alternated with 24-hour recovery periods at 21 degrees Celsius. It was found to increase the indel rates of Cas9 and LbCas12a. Quantification of the genome editing efficiency was conducted using visual mutant phenotyping, and genotyping by Sanger sequencing. The scientists were also able to develop a support protocol to efficiently clone a CRISPR expression vector using only one step.

The assay allows scientists to increase CRISPR-induced mutations using a low-cost setup in plant research.

Read Current Protocols for more details.

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