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.�As a proof of concept,�CsPDS and/or�CsLOB1 are chosen for targeting through transient expression of Cas9-sgRNA or Cpf1-crRNA via Xcc-facilitated agroinfiltration. Recently, we employed CRISPR-LbCpf1, derived from�Lachnospiraceae bacterium�ND2006, to edit the citrus genome. First, LbCpf1 was successfully used to modify Duncan�CsPDS via Xcc-facilitated agroinfiltration. Next, GFP-p1380N-35S-LbCpf1-crRNA-lobp and GFP-p1380N-Yao-LbCpf1-crRNA-lobp were constructed to edit the PthA4 effector binding elements in the�CsLOB1 promoter (EBEPthA4-CsLOBP) in transgenic Duncan grapefruit. Totally, seven GFP-p1380N-35S-LbCpf1-crRNA-lobp-transformed Duncan plants were generated, designated as #D35s1 to #D35s7, and ten GFP-p1380N-Yao-LbCpf1-crRNA-lobp-transformed Duncan plants were created, designated as #DYao1 to #DYao10. LbCpf1-directed EBEPthA4-CsLOBP modification was observed in three 35S-LbCpf1-transformed Duncan (#D35s1, #D35s4 and #D35s7). However, no LbCpf1-mediated indels were observed in the Yao-LbCpf1-transformed plants. Importantly, transgenic line #D35s4, containing the highest mutation rate, alleviates Xcc.pthA4:dCsLOB1.4 infection.�Therefore, CRISPR-LbCpf1 can be readily used as a powerful tool for citrus genome editing. We have also made progress regarding use other Cas proteins.�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 promotors� 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. We are also filing patent for this technology.��