Increasing Gene Editing Efficiency in Citrus through Transient Expression of CRISPR-Cas9 Proteins and Heat Treatments

The CRISPR-Cas9 system has been used to edit genes in several citrus genotypes. Early demonstrations of successful genome editing in citrus were based on targeting the phytoene desaturase (PDS) gene. Despite of these early successes, the gene editing efficiency of this system in citrus needed to be increased substantially for routine use. In this study, we demonstrated enhanced gene editing efficiencies in two citrus genotypes (Carrizo citrange and Duncan grapefruit) by transient expression of two Cas9 proteins [SpCas9 and high fidelity spCas9 (HF-spCas9)] and heat treatments. Mutations were detected in citrus leaf and callus tissues 96 and 144 hours after Agrobacterium inoculation. Heat treatment at 37 ËšC for 48 hours increased mutation efficiency by several folds. Transient expression of Cas9 proteins and gRNAs in citrus tissues will be beneficial in selecting gRNAs with higher editing efficiencies for producing stable genome edited citrus plants.