Heat Stress Induced Potato virus X-mediated CRISPR/Cas9 Genome Editing in Nicotiana benthamiana

Heat Stress Induced Potato virus X-mediated CRISPR/Cas9 Genome Editing in Nicotiana benthamiana

Targeted genome editing using CRISPR/Cas nucleases has become the standard approach for creating mutant plants. Significant progress has been made to enhance the editing efficiencies through optimizing CRISPR/Cas expression, including applying heat stress. In this study, we used heat stress to enhance the Potato virus X (PVX)-mediated CRISPR/Cas9 mutagenesis in Nicotiana benthamiana. We show that heat stress at 4-5 days after PVX inoculation effectively increases the mutagenesis efficiency of Cas9 nuclease. We observed up to a 5-8% increase in mutation efficiency depending on the sgRNA construct when heat stress is applied to the pPVX-Cas9::sgRNA infiltrated samples. Furthermore, analysis of the effect of the heat stress on the pattern of mutation types in the target gene regions showed no obvious changes in CRISPR/Cas9 induced mutagenesis pattern between heat stress treated and no heat stress treated samples. Overall, our experiments demonstrate that heat stress treatment at the optimal time after viral inoculation is most effective in increasing the PVX-mediated CRISPR/Cas9 editing efficiency in plants.