Heat stress increases mutation efficiency mediated by CRISPR/Cas9 in citrus

The CRISPR/Cas9 system has shown great promise in engineering targeted mutations in a genome. The efficiency of Cas9-mediated genome editing is temperature sensitive. A high-temperature regime can increase the mutation efficiency induced by the CRISPR/Cas9 system in many plant species. However, a heat stress treatment has not been applied during the tissue culture process in citrus. To develop an efficient heat stress regime to improve the efficiency of CRISPR/Cas9-mediated targeted mutagenesis, three and five cycles of heat stress treatments were used during callus induction in citrus. The results showed that the heat stress treatment with three cycles of 24 h at 37 °C, followed by 24 h at 26 °C, increased the mutation efficiency by 11.6% compared with no heat stress treatment, and that five cycles of heat stress treatment were optimal, from which 50% mutants had a 100% mutation rate. The mutation profiles of Cas9 at 28 °C for 10 d and 37 °C for three or five cycles were similar, indicating that heat stress treatment did not affect the non-homologous end joining repair pathway. No detectable off-target mutation was detected in the potential off-target sites with four nucleotide mismatches compared with the designed on-target site. This study demonstrated that five cycles of heat stress treatment during callus induction could efficiently increase the mutation efficiency mediated by the CRISPR/Cas9 system without observable negative effects, and provided an efficient Cas9-mediated citrus genome editing system to produce mutants with a high mutation rate.