Performing Department
(N/A)
Non Technical Summary
CRISPR is a powerful tool for breeding new HLB-resistant citrus varieties, but has to be preceded by identifying specific gene-editing targets. Previous NIFA funded research identified CLas effector proteins and interacting citrus genes, however no research has been performed on editing multiple of these discovered target genes simultaneously to develop fully-HLB-resistant varieties.Soilcea has previously edited three genes individually that have shown improved resistance or tolerance to HLB. Given HLB's complex disease pathway, we will multiplex edit these three identified and proven HLB susceptibility genes simultaneously to develop new varieties with more durable resistance. For some varieties, we will also edit a gene for canker resistance.This proposal will use CRISPR multiplexing and protoplast transfection to target three or four previously identified and proven resistance genes simultaneously to develop non-transgenic, HLB and canker-resistant citrus varieties.This project addresses ECDRE's first priority need: "Development of commercial citrus varieties . . . for both fresh and processed markets with genetic resistance . . . to HLB using . . . gene editing." This project will work with sweet orange, and mandarin varieties.The first activity is multiplexed CRISPR-editing of three promising HLB SDE-interacting genes and one canker effector-interacting gene simultaneously, and then generation of non-transgenic, CRISPR-edited citrus varieties. The second activity is field trials to screen new CRISPR-edited varieties for disease resistance and to assess the varieties for their horticultural traitsWe anticipate developing non-GMO, HLB and canker-resistant citrus trees that will be sold to growers.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Goals / Objectives
Soilcea in conjunction with the University of Florida will employ CRISPR-multiplexing to edit citrus protoplast to develop Huanglongbing ("HLB")-resistant sweet orange and mandarin varieties. Clustered Regularly Interspaced Short Palindromic Repeats ("CRISPR")-mediated editing is the most powerful tool for breeding new Huanglongbing ("HLB") resistant cultivars without possessing conventional GMO issues. The promise of CRISPR, however, has to be preceded by identification/selection of specific targets that are going to be edited by this genome engineering tool.In previous USDA NIFA funded studies, promising HLB target susceptibility genes were discovered. Soilcea exclusively licensed those target genes and a canker susceptibility gene from the University of Florida, and has shown that editing three genes individually provides tolerance or improved resistance to HLB. When editing two of the genes simultaneously, those trees exhibit increased tolerance and resistance. Soilcea has developed non-transgenic Valencia and Hamlin varieties with improved resistance to HLB targeting one and two of the priority HLB-susceptibility genes, and Carrizo rootstock varieties targeting three of those priority target genes. This proposal will use CRISPR multiplexing and protoplast transfection to target three identified and proven HLB-susceptibility genes in sweet orange mandarin varieties. For some varieties, we will also simultaneously edit a canker resistance gene.Successful accomplishment of this three-year project will generate non-GMO, HLB-resistant citrus varieties. At the end of the project, we will have produced CRISPR-edited citrus trees that have unique combinations of CRISPR edits to promising HLB-susceptibility genes. These genes are involved in disrupting Candidatus Liberibacter asiaticus effector proteins that facilitate HLB infection. These CRISPR-edited trees will be developed using polyethylene glycol (PEG)-mediated transfection of isolated protoplasts. Specifically, Soilcea has Hamlin and Valencia callus lines ready for transfection, and our collaborator's lab, Dr. Ahmad Omar, has W. Murcott, Nova Mandarin, and OLL-8 lines ready for transfection, and is working on developing callus for other lines. Time permitting, we may work with other mandarin varieties and sweet orange varieties when callus lines are available. Our lab has been successful in developing non-GMO, CRISPR-edited Valencia and Hamlin varieties using this protoplast transfection technique to edit two genes simultaneously. The new varieties will then be clonally propagated and prepared for field trials to evaluate their level of HLB-resistance in a commercial setting.The deliverables from this project will be HLB-resistant citrus trees developed from CRISPR precision breeding. There will be five milestones during this project: (1) construction of 7 distinct multiplex CRISPR vectors targeting three and four genes each; (2) 25 protoplast transfections per construct with at least one sweet orange variety and one mandarin variety; (3) material will be put through the regeneration process and any resulting plantlets will be screened for CRISPR edits as well as confirmation of no off target mutations or foreign DNA integrations; (4) edited plants will undergo clonal propagation and evaluation of resistance through field trials; and (5) delivery of plants to relevant cleanup agencies and then nurseries for commercial propagation.
Project Methods
1-1. Plasmid construction of multiplex CRISPR constructsAs mentioned above, achieving a high level of HLB resistance will likely require edits in multiple gene targets, and we already have three identified and proven genes that confer HLB resistance or tolerance on their own, and a proven canker-resistance gene. Due to variation in gene sequences between Citrus species, new sgRNAs will be designed to target the specific sequences of our four target genes in the citrus varieties that we will work with during this project.For multiplexed CRISPR gene editing, we have generated a new tRNA-based multiplex CRISPR plasmid vector, pSMulti-Non-Transgenic (pSMNT), modified from the published multiplex CRISPR system (Huang et al., 2020), which will be used to synthesize the multiplexed sgRNA sequences and ligate them into the pSMNT vector through Golden Gate cloning. For each sgRNA, a pair of annealed oligos comprising the 20 bp sgRNA sequence as well as 4 bp complementary overhangs for ligation will be synthesized by Eurofins Genomics (Louisville, KY). Each set of sgRNA annealed oligos will be cloned into an intermediate plasmid through simple restriction enzyme digestion and ligation. The intermediate plasmids will then be used with our empty pSMNT vector for Golden Gate cloning to assemble the final plasmids for our experiments. This cloning system consists of a modular design enabling the construction of different sgRNA combinations to ensure we can easily and efficiently design and clone any combination of our gene targets to use for these experiments.Recently, Soilcea has started to use pre-assembled CRISPR ribonucleoprotein (RNP) complexes during transfection experiments. This technique has potential to increase gene editing efficiency as well as mitigate the risk of any DNA integrations. Soilcea plans to incorporate both the plasmid-based transfection method which has proven successful as well as RNP-based experiments to ensure that we can generate HLB-resistant plants as quickly and efficiently as possible.1-2. Generation of callus and callus maintenance for commercially important varietiesWe will follow the protocol outlined by Omar et al. (2016) to establish callus lines from sweet orange and mandarin varieties. Immature fruits will be collected annually from the selected cultivars.1-3. Protoplast transfectionFor the non-transgenic gene editing, Soilcea has designed a non-transgenic multiplex-compatible Cas9-sgRNA vector system (pSMNT) (Figure 4). This approach increases the likelihood that plants will not be transgenic because this approach does not utilize Agrobacterium tumefaciens, and there are no DNA-based mechanisms present to facilitate insertion into the genome. Soilcea has confirmed no foreign DNA present in protoplast regenerants in previous experiments through whole-genome sequencing. We will also use RNPs to ensure no integration of foreign material. For transfection, protoplasts will be isolated from suspension cultures and the Cas9-multiplexed sgRNA plasmid constructs or RNPs will be delivered into protoplast cells (Guo et al., 2005; Omar & Grosser, 2007; Huang et al., 2020).1-4. Protoplast regeneration of non-transgenic CRISPR-edited citrus varietiesThe next step in the proposal will be to regenerate transfected cells. The transfected cells will be regenerated using combinations of liquid and solid media with varying ratios of auxin and cytokinin hormones (Omar & Grosser, 2007).All citrus plants that regenerate will be assayed for successful editing. Leaf tissue will be collected for DNA extraction and then used in PCR with primers that flank the Cas9 cut site. The PCR amplified sequences will then be Sanger sequenced to identify edits in the target locus. Plants that carry the desired edit will be retested, and then whole-genome sequenced for confirmation and off-target analysis and then clonally propagated.2-1. Clonal PropagationThe replicates for this experiment will be obtained by clonal propagation, micrografting, and traditional grafting. When regenerating scion material, any branches that begin to grow will be excised and clonally propagated. Shoots products from those stem segments will be micrografted onto rootstocks in culture. Plants will be transplanted into soil, acclimated, and grown in a greenhouse before handing off for field testing. Once plants get to a sufficient size, then we will perform traditional grafting when necessary to increase the number of replicates. We also plan to provide material early on to nurseries, such as Agromillora, to begin large scale propagation.2-2. Plant CRISPR-edited citrus varietiesThe purpose of this field trial will be to test the level of HLB tolerance/resistance in CRISPR-edited citrus plants. We have designed these field trials by following the Plant Improvement Germplasm Evaluation Guidelines (CRDF, 2018). The trial will consist of assessing HLB symptoms in CRISPR-edited scions grafted onto commercial rootstocks and already developed CRISPR-edited Carrizo rootstocks. This will assess whether resistance to HLB requires only a CRISPR-edited scion or a combination of an edited scion and rootstock. The trial will start out with 1 acre of citrus plants at a density of about 300 plants per acre, which follows the 2022-2023 citrus grove design recommendations (Singerman et al., 2022). Our CRISPR-edited plants will be tested against wild-type controls. For each construct and the control, several lines will be assessed with 3 biological reps of each tree planted consecutively within a row and at least 3 technical replicates laid out in a randomized complete block design (Albrecht and Bowman, 2012). All plants will be grown together in a randomized arrangement in the greenhouse prior to planting in the field.Before the start of the trial, all scions and rootstocks will be screened for their CLas titer levels using a qPCR-based approach. The grafted plants will then go through an 8-week period of high exposure to psyllids in an enclosed hoop house to initiate HLB infection before planting the trees for the trial to compensate for any lack of disease pressure (Grosser et al., 2014).2-3. qPCR for CLas titer measurementThe screening process will follow the Plant Improvement Germplasm Evaluation Guidelines approved by researchers from Arizona, California, Florida and Texas (CRDF, 2018). Three leaf samples from the same locations on each tree will be collected at 6 month intervals. The leaf samples will be stored at -80°C before genomic DNA and RNA is extracted from the midrib. The CLas titer level will be determined by DNA and, in some cases, RNA-based qPCR amplifying both a conserved sequence from the CLas flagellum as well as a conserved citrus sequence as an internal control.2-4. Visual observation of HLB symptomsThe following tree measurements will be taken at the beginning and end of the trial and every 6 months in between: trunk diameter, canopy diameter/volume, and tree height. Foliar disease ratings, including photographic documentation, will be conducted each spring and fall. Percentage of leaves with symptoms will be scored on a 1-5 scale. General canopy thickness and color will also be rated 1-5. All of these measurements will be used to give an overall HLB symptom severity score of 0-5. At the same time as foliar disease ratings, leaves and fibrous roots will be randomly collected for qPCR detection of CLas. Additionally, nutrient concentrations will be measured in the summer in 4-6 month old spring flush leaves. Finally, if the citrus trees are producing fruit, then fruit yield, size, weight, and juice quality will also be measured and recorded.