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ARS Home » Northeast Area » Kearneysville, West Virginia » Appalachian Fruit Research Laboratory » Innovative Fruit Production, Improvement, and Protection » Research » Research Project #439596

Research Project: Phloem Targeted Multiplexed Gene Editing for Enhanced Control of Huanglongbing in Citrus

Location: Innovative Fruit Production, Improvement, and Protection

Project Number: 8080-21000-029-024-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Oct 1, 2020
End Date: Aug 31, 2023

The objectives for this project are: 1) Development of multiplexed gene editing and gene activation methods in citrus; 2) Identification and validation of phloem and defense promoter specific sequence motifs for use in transcriptome reprogramming; and 3) Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses.

Objective 1: The first task will be to establish citrus protoplast transformation systems. For protoplast production, embryogenic cell lines from the sweet orange varieties Hamlin and Valencia will be used for the described gene editing transformations. Successfully prepared protoplasts will be transformed with a green fluorescent protein (GFP) reporter constructs and tested after two days to determine transformation efficiency. Our second task will be to develop a multiplexed Cas12a system for promoter editing. Our recently developed multiplexed Cas12a gene editing system and Cas12a RNP system can be translated into the citrus protoplasts. Our first strategy is to use these systems for promoter editing to impact target gene expression. Our second strategy is to conduct Cas12a-mediated homology-directed repair (HDR) to insert cis-elements to impact gene expression. Our third task will be to develop CRISPR-Act3.0 for tissue-specific activation in phloem. To assemble the multiplexed CRISPR-Act3.0 gene activation systems, the gRNAs will be designed as oligos, and then annealed and subsequently cloned into gRNA expression vectors. The resulting vectors will be used for citrus protoplast transformation. Objective 2: This task will be include silico identification of target gene promoter sequences. The ARS scientist will lead specific studies to mine the promoter domains of defense associated genes found to be differentially regulated within CLas infected tolerant citrus, as well as the promoters from identified phloem specific genes. The second task will be expression analysis and validation of identified target genes. Efforts here will confirm and identify the expression and tissue localization of selected differentially expressed genes (DEGs). Defense associated genes that display differential expression in resistant citrus and are expressed in the phloem would be a priority for transcriptional reprogramming. Objective 3: Validation of gene editing systems for the targeted transcriptional reprogramming of host defense responses. The first task will be qPCR analysis of promoter edited protoplasts and calli for and regenerated germplasm. Studies here will examine the efficiency of gene editing systems to modify the activity and expression of targeted genes for use in CLas resistance screening. Specifically, edited calli and subsequent regenerated germplasm will be examined for the stability of edited sequences and effect on target gene transcriptional reprogramming. The second task will be regeneration and in situ expression analysis of promoter edited calli and germplasm. Citrus germplasm regenerated from transformed and edited protoplasts will be assessed for the impact of the designed promoter edits on target gene expression.