The purpose of this project is to optimize the CRISPR technology for citrus genome editing. This study is related to the CRDF RMC-18 Research Priorities 4AB. We are optimizing the CRISPR-Cas9 technology in citrus genome editing by conducting the following three objectives: Objective 1. Expanding the toolbox of citrus genome editing. In this study, we will adapt StCas9, NmCas9, AsCpf1, FnCpf1 and LbCpf1 on genome modification of citrus. Lately, we have shown CRISPR-Cpf1 can be readily used as a powerful tool for citrus genome editing. One manuscript entitled CRISPR-LbCas12a-mediated modification of citrus has been published on Plant Biotechnol J.Objective 2. Optimization of the CRISPR-Cas mediated genome editing of citrus. In this study, we will first test different promoters in driving expression of Cas9 and Cpf1. We have identified one optimized promoter which showed higher efficacy in driving gene expression in citrus than 35S promoter and Arabidopsis U6 promoter. We are further characterizing the promoter and test its efficacy in driving sgRNA and in genome editing of citrus. We have also developed a method to increase the transient expression efficiency. Objective 3. Optimization of the CRISPR technology to generate foreign DNA free genome editing in citrus. We have conducted transient expression of Cas9/sgRNA plasmid and Cas9 protein/sgRNA ribonucleoprotein complex in citrus protoplast. The plasmid-transformed protoplast has 1.7% editing efficiency, and the RNP-transformed samples have approximately 3.4% efficiency. The genome modified protoplast cells are undergoing regeneration. We aim to increase the efficacy to over 20% and eventually generate non-transgenic genome modified citrus. One manuscript is under preparation. One patent has been filed on the CRISPR-Cas mediated genome editing of citrus.