Document Type

Thesis - Open Access

Award Date

2022

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Yang Yen

Keywords

BSMV, By Pass Tissue Culture, CRISPR/Cas, Delivery of Cas Enzyme, Plant Gene Editing

Abstract

CRISPR/Cas system is a powerful gene-editing tool that has revolutionized gene editing in many ways. However, the current delivery methods for using CRISPR/Cas system inn plants have some drawbacks that have greatly limited their routine application in plant research and improvement. Specifically, the methods heavily rely on either genetic transformation for delivering Cas and/or its gRNA coding sequences into the targeted cells or tissue culture, or both for recovering the whole edited plants. To overcome these drawbacks, we have developed a simple, easy to use yet effective system for CRISPR/Cas gene editing in plants that can bypass genetic transformation and tissue culture. A viral vehicle has been constructed out of the gamma RNA of barley stripe mosaic virus (BSMV) that is able to simultaneously deliver and express both Casφ2 and its gRNA genes. Simultaneous expression of Casφ2 and reporter gene mCherry (in the place of sgRNA) were observed in both the inoculated and non-inoculated leaves of the inoculated Nicotiana benthamiana plants. Juice collected from the inoculated tobacco leaves, which contains the BSMV vector, successfully inoculated the viral vector into Arabidopsis, maize, rye and wheat plants via seed imbibition. We also demonstrated that stable Casφ2 gene can be expressed in Arabidopsis, maize, rye, wheat, and tobacco. Our data showed that this BSMV deliver/expression system is capable to bypass genetic transformation and tissue culture for CRISPR/Cas gene editing in plants, and it is simple to use. Further research is needed to study how widely this CRISPR/Casφ system can be used in plant gene editing. The system developed in this project provides a promising tool that has a great potential to revolutionize molecular precision breeding in crops.

Number of Pages

162

Publisher

South Dakota State University

Available for download on Sunday, August 31, 2025

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