Non Technical Summary
Previous studies demonstrate that citrus HLB is a pathogen-triggered immune disease. CLas stimulates a systemic and chronic immune response in citrus phloem including reactive oxygen species (ROS) production and callose deposition, which causes systemic phloem cell death and subsequent HLB disease symptoms. The goal of the project is to generate transgene-free HLB resistant/tolerant citrus varieties using the cutting-edge precision genome editing technology. The central hypothesis is HLB can be controlled by mitigating CLas-triggered ROS through knocking out the upstream open reading frames (uORFs) of genes encoding antioxidant enzymes and antioxidants. Because uORFs usually repress the translation of primary ORFs, editing of the uORFs is expected to increase levels of antioxidant enzymes and antioxidants to mitigate CLas-triggered ROS production, phloem cell death and HLB symptoms. Our approaches are novel by utilizing the most advanced transgene-free citrus genome editing techniques and precision genome editing techniques (i.e., base editing and prime editing). Four objectives are proposed: 1) Develop feasible and efficient transgene-free precision genome editing systems in citrus. 2) Transgene-free genome editing of the uORFs of genes encoding antioxidant enzymes and antioxidants. 3) Evaluate the genome-edited citrus varieties for HLB resistance/tolerance, and other horticultural traits. 4) Deliver HLB management approaches/products through extension and outreach. Transgene-free HLB resistant/tolerant citrus varieties with suitable horticultural traits will provide the most effective, environmentally friendly and economic approach for HLB control.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0999	2020	50%
212	0999	3100	30%
212	0999	1040	20%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1040 - Molecular biology; 2020 - Engineering; 3100 - Management;

Goals / Objectives
The goal of the project is to generate transgene-free HLB resistant/tolerant citrus varieties using the cutting-edge precision genome editing technology.Four objectives are proposed: 1) Develop feasible and efficient transgene-free precision genome editing systems in citrus. 2) Transgene-free genome editing of the uORFs of genes encoding antioxidant enzymes and antioxidants. 3) Evaluate the genome-edited citrus varieties for HLB resistance/tolerance, and other horticultural traits. 4) Deliver HLB management approaches/products through extension and outreach.

Project Methods
Objective 1. Develop feasible and efficient transgene-free precision genome editing systems in citrus1.1. Adenine base editor (ABE) RNP transformation of embryogenic protoplasts Protein expression and purification of ABE. ABE expression and purification will be conducted. Briefly, ABE8e containing an N-terminal His6-tag will be cloned into a pBR322 plasmid. The recombinant proteins will be expressed inE. colistrain BL21 Rosetta 2 (DE3) (EMD Biosciences) and purified.Testing editing efficacy of ABE. To characterize the editing efficacy of ABE, ABE RNP will be used to transform embryogenic C. sinensis cv. Hamlin protoplasts using the PEG method. The RNP-transformed embryogenic citrus protoplasts will be used for callus induction and plant regeneration. PCR amplification, Sanger sequencing, and amplicon deep sequencing analysis of RNP transformed protoplasts for the target site will be conducted to analyze the efficacy of A⋅T to G⋅C base conversion in the uORF start codon of the four target genes.1.2. Prime editor RNP based transformation of embryogenic protoplasts. The prime editor PE5max expression and purification will be conducted. The engineered pegRNAs (epegRNAs) will be designed and synthesized to contain an RT template encoding the conversion of the uORF initiation codon ATG to CTG and structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. A nicking guide RNA (ngRNA) will be designed and synthesized that nicks the non-editing strand and favors edits during mismatch repair. Transformation of embryogenic protoplasts with PE5max RNP and testing of the editing efficacy will be conducted as described in Objective 1.1.1.3. Agrobacterium-mediated co-editing for ABE-CBE, PE5max, and CBE via epicotyl transformation. We will construct the binary vector ABE-CBE-GFP. GFP will be used for selecting transgene-free (green fluorescence absent) transformants; CBE/gRNA will be used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron as a positive selection marker, which has no negative effects on plant phenotypes; ABE/gRNA will be used for conversion of the uORF initiation codon ATG to GTG or ACG of the four target genes. The ABE-CBE-GFP binary vector will be used for Agrobacterium-mediated transformation of epicotyl tissues of C. sinensis cv. Hamlin. The regenerated shoots which are negative for green fluorescent and resistant against chlorsulfuron will be tested for editing efficacy of the uORF initiation codon ATG based on PCR and sequencing and determined whether they are transgene-free by PCR approach using primers specific for GFP, ABE, and CBE.Similarly, we will construct binary vector PE5max-GFP with PE5max for multiplex prime editing ALS and target genes. For the CBE-GFP construct, we will use the tRNA-based multiplex system with one gRNA targeting the ALS gene and the other targeting the uORF start codon ATG to convert it to ATA.Objective 2. Transgene-free genome editing of uORFs of genes encoding antioxidant enzymes and antioxidantsTo edit the uORFs of target genes, we will transform embryogenic protoplasts of C. sinensis cv. Hamlin using base editor RNP, or prime editor PE5max RNP, or transform epicotyl via Agrobacterium-mediated delivery of ABE-CBE, PE5max or CBE as described in objective 1. To change the start codon of uORFs of antioxidant enzyme genes and genes involved in antioxidant biosynthesis, we will conduct genome editing of the uORF of each individual gene. In addition, we will conduct simultaneous editing the uORFs of SOD/APX, or SOD/GPX using the tRNA-based multiplex system to carry one gRNA targeting SOD and another gRNA targeting APX or GPX.The transfected embryogenic protoplasts will undergo callus induction and plant regeneration. The shoots will be micro-grafted on Carrizo rootstock for further confirmation. The transformed epicotyls will undergo plant regeneration; chlorsulfuron-resistant and green fluorescence absent shoots will be micro-grafted on Carrizo rootstock for further testing.To screen for genome edited plants, we will PCR-amplify the relevant regions and sequence the amplicons for individual target. It is expected that editing of either a single allele or both alleles can increase the expression of antioxidant enzyme genes and genes involved in antioxidant biosynthesis even though homozygous/biallelic mutants might have higher expression levels. Thus, heterozygous, homozygous or biallelic mutants will all be kept for downstream evaluation. For the edited lines, we will analyze the extent of putative off-target mutagenesis. The off-target analysis will be conducted by PCR amplification and sequencing of putative off-target sites or using deep sequencing. In addition, whole genome sequencing of all edited lines will be conducted using the Illumina NovaSeq 6000 platform for further analysis of genome editing of target sites and off-target mutations. Each confirmed mutant line without off-target mutations will be propagated to more than 50 plants on Swingle citrumelo rootstock by grafting for greenhouse assays and field trials.Objective 3. Evaluate the genome-edited citrus varieties for HLB resistance/tolerance, tree growth and development, and other horticultural traits3.1. Greenhouse assays. The genome-edited lines (20 plants/line) and wild type (20 plants) of 12-month-old will be inoculated with CLas via grafting in greenhouse. In addition, the genome-edited lines (5 plants/line) and wild type plants (5 plants) of 12-month-old will not be inoculated with CLas and be used as controls. We will quantify CLas in the inoculated plants with qPCR. From the confirmed CLas-positive plants, we will select six edited plants/line and six wild type plants that yield similar Ct values for further testing with one tree as a biological replicate. We will measure ROS production and cell death of phloem tissues in CLas-positive or CLas-free genome-edited lines and wild-type plants in an interval of three months within a duration of 12 months. We will monitor HLB symptoms monthly and investigate tree growth (trunk diameter, height, and canopy) annually. In addition to monitoring HLB symptoms, we will also conduct analyses of callose deposition and starch accumulation, which are commonly observed in HLB symptomatic trees every three months.3.3.2. Field trials. The genome-edited lines (20 plants/line) and wild type (20 plants) of 12-month-old will be planted in a citrus grove in Citrus Research and Education Center, University of Florida. We will quantify CLas titers every three months, monitor the disease severity and incidence of HLB of the edited lines and wild type control plants in the natural environment. We will investigate ROS, callose deposition around sieve pores, starch accumulation and phloem cell death every three months once the trees become infected with CLas. To test whether the genome edited plants are affected in normal citrus growth, we will evaluate the size of the seedlings (height and trunk diameter).Objective 4. Extension and outreachWe will organize workshops, grower meetings, field day events regarding transgene-free citrus genome editing and using non-transgenic genome edited citrus varieties to control HLB. Information will be published in industry magazines such as "Citrus Industry", and Cooperative Extension newsletters, and disseminated to the stakeholders at extension events such as Citrus Expo.