Location: Grape Genetics Research Unit (GGRU)
Project Number: 8060-21220-008-008-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 15, 2022
End Date: Sep 14, 2025
Objective:
Develop nano-based methods for delivering DNAs, RNAs and/or proteins into plant cells for modifying grapevine genes non-transgenically.
Gene editing technologies have successfully been applied to gene modifications in plants, but most of the work was done through transgenic methods. Several non-transgenic editing approaches have actively been pursued, including in vitro ribonucleoprotein (RNP) transfection through electroporation, magnetic-mediated transfection, transient expression of transgenes without DNA integration, and uses of virus-like particles, bacterial secretory systems, and cationic polymers and/or nanomaterials such as carbon nanotube (CNT) for Cas9/guide RNA delivery. Unfortunately, none of them have been proven working effectively and efficiently. Among many challenges, plant cell walls present a significant barrier to the delivery process, especially when the molecular sizes of DNAs/RNAs/proteins to be delivered are big. Rapid degradation of the delivered DNAs, RNAs and proteins in cells adds additional challenges. In this collaborative work, we plan to evaluate if DNA hydrogel technologies, such as the one recently developed by the Cooperator (PI of Cornell University), would help resolve the problem. DNA hydrogels are composed of only DNA and water molecules. These DNA hydrogels can be used as genetic templates to carry out “the central dogma of the molecular biology” – producing functional proteins in a cell-free fashion. DNA hydrogels consist of a three-dimensional network of genes that are covalently crosslinked (ligated). In other words, genes are the matrix of the gel. Thus, physically, it is a gel but biologically it functions as a collection of functional genes with no possibility to integrate the gel into any genomes. Furthermore, a DNA hydrogel provides better protection of its cargos (DNAs/RNAs/proteins) from degradation. These unique properties may offer us an unprecedented opportunity to overcome the aforementioned challenges in transfecting plant cells.
Approach:
The initial duration of this collaborative work is two years;
Year 1: Concept evaluation. A GFP-DNA-gel will be constructed and introduced into plant cells through particle bombardment to test the gel delivery process and GFP expression. Gold nanoparticles (AuNP) will be used as the core and coated with the GFP-gel. The coated AuNP will be bombarded into embryogenic callus cells of the wine grape ‘Chardonnay’ and/or table grape ‘Thompson Seedless’. The delivery efficiency will be quantified and assessed by the percentage of intake cells along with the overall GFP expression. Optimizations include but are not limited to the size of the AuNP core, the thickness of the outer gel layer, the concentration of the cargo gene, and different bombardment conditions. If successful, an expression cassette containing CRISPR-Cas9 and GFP gRNAs will be incorporated into a GFP gel. The GFP gel carrying Cas9 editing components (abbreviated as GFP-Cas9) will be introduced into plant cells through bombardment as described above. The GFP editing efficiency will be determined by comparing GFP signals observed in the callus cells transformed with GPF-Cas9 and GFP gels, respectively.
Protein co-delivery will also be explored by using an RFP protein pre-loaded into the GFP-gel. The delivery efficiencies will be quantified and assessed by the percentage of intake cells with RFP signal. If successful, CRISPR-Cas9 proteins and gRNAs will be pre-loaded into the GFP gel. The GFP editing efficiency will be determined similarly as described above.
Year 2: Editing grape genes. Pending on the success of achieving first year’s goals, we will construct CRISPR gels with either Cas9 enzyme and gRNAs pre-loaded or expression cassettes of Cas9 gene and gRNAs incorporated into the gel. Several gRNAs targeting specific grapevine genes will be tested. Initial test will focus on the grape PDS gene for which extensive editing experience has been acquired by the ARS researchers. The PDS editing efficiencies will be determined through high throughput sequencing of 3–5-month-old transformed callus, by evaluating albino phenotypes in regenerated vines from the transformed callus, and/or by subsequent molecular analysis of albino vines. If the PDS editing is successful with an acceptable editing efficiency, gRNAs for targeting other grape genes of significant biological and/or economic relevance will be attempted.
