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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Research Project #442597

Research Project: Developing New Methodologies to Obtain High- Throughput, Transgene- Free Gene Edited Microvine

Location: Crop Improvement and Genetics Research

Project Number: 2030-21220-003-010-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2022
End Date: Aug 31, 2025

The proposed research is broken down into two main projects that are to i) optimizing a viral-based transgene-free gene editing methodology with intact microvine cells, and ii) developing a transgenic microvine line suitable for generating high-throughput mutant collections. From the first project, we have defined three objectives that are: Objective 1: We will compare the replication efficacy of a Red-Blotch derived geminiviral replicon to that developed by Olivares et al., 2021 by measuring the expression of green flourescent protein (GFP) following a vacuum-agrobacterium infiltration assay using embryogenic cells of the 04C023V0004 microvine genotype, also called V4. Objective 2: We will compare the editing efficiency rate of an intron-based single transcript unit (STU) to a regular STU with two leading Sequence-Specific nucleases (SSNs), the Cas9 and Cas12a system, designed to disrupt the GFP expression of V4 microvine transgenic plants following a stable Agromediated-transformation. Objective 3: We will test the editing mutagenesis rate on eGFP in V4 eGFP-expressing lines following a transiently expressed GBRV-derived Geminiviral replicon. We will then evaluate the absence of T-DNA insertion by next generation sequencing (NGS) or restriction fragment length polymorphism (RFLP) from the successfully edited lines.

Developing an efficient CRISPR editing technology that produces transgene-free grapevine is paramount for the grape industry. Several approaches are currently being pursued in grapevine that primarily uses the RiboNucleoProtein as a material to be delivered including biolistic, transfection to protoplasts, and nanomaterial-complexation. However, the inherent limitation of each technology suggests a need for additional editing toolkits to deliver gene-editing reagents with higher efficiency for generating transgene-free edited plants. A DNA-based delivery strategy of a self-replicating viral system delivered to the intact plant regenerable cells will be examined. We propose to develop a novel Geminiviral replicon using a Grape Red Blotch Virus as an editing delivery system. The research will focus on several milestones that include i) the comparison of the replication efficacy of the current mastrevirus geminivirus replicon (GVR) to a newly developed GVR based containing the self-replication machinery of the GRBV, ii) the evaluation of the Intron-Based Single Transcript Unit for higher efficiency in grapevine cells with two types of Cas protein systems (Cas9 and Cas12a) via conventional Agrobacterium-mediated transformation, iii) the determination of the efficiency editing rate of a transiently expressed Red-blotch-based GVR containing the gene-editing reagents via Agroinfiltration of embryogenic cells. Like many crops, two significant bottlenecks have hampered the generation of mutant collection in grapevine, the low plant regeneration rate from genetic transformation and the lack of high throughput systems for targeted mutagenesis. We propose to tackle these two hurdles by developing a unique genetic construct of a microvine line that will be used for high throughput mutagenesis. Along with GFP and Hygromycin resistance selection marker, the construct will be a growth regulating factor (GRF)-GIF gene fusion shown to improve plant regeneration from tissue culture. The proposed research will examine i) the importance of conditional expression of the GRF-GIF in the amount of plant regeneration after Agromediated-transformation, ii) the efficacy of Cas12a versus Cas9, and the value of Intron-based Single Transcript Unit versus Standard Transcript Unit in terms of editing efficiency, and iii) the utilization of cell-penetrating peptides to deliver the guide RNA to intact regenerable microvine cells as previously described in other plant models. The overall objective is to create a transgenic line that stably expresses an active Cas enzyme and that is available for the grapevine community to rapidly and efficiently generate targeted mutants via exogenous delivery of a guide ribonucleic acid (RNA).