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Shan, Shengchen [1], Yang, Bing [2], Soltis, Douglas E. [3], Soltis, Pamela S. [4], Barbazuk, W. Brad [5].

Developing CRISPR systems in non-genetic plant models: from plasmid design to transgenic plant.

CRISPR is a powerful tool for genome editing. The past eight years have seen the rapid growth of CRISPR studies in both basic research and practical applications. However, most of the genome-edited plants are genetic models, such as Arabidopsis and major crops. Developing an efficient genome editing system in non-genetic model plant systems is challenging, but holds enormous potential in plant science. In this workshop, based on our own experience in utilizing CRISPR, we will provide a step-by-step guide of how to apply CRISPR in any plant system: from plasmid design to transient assays and the generation of stable transgenics. In addition, we will provide resources and discuss the considerations of developing CRISPR in plants of interest to the participants, based on polling prior to the workshop. Assembly of the plasmid (i.e., the CRISPR construct) is the initial step of plant transformation. The expression of foreign genes in a plant genome is related to the efficiency of the genome editing system, and the choice of the sgRNA (single-guide RNA) sequence determines the gene knock-out efficiency and the off-target effects. In this workshop, we will introduce and compare various CRISPR constructs with different promoters driving the expression of the Cas9 gene and sgRNAs. In addition, we will discuss the process of sgRNA design for a candidate gene. Transient assay is a time-saving approach to examine the genome editing efficiency of a CRISPR system in non-genetic model plants. We will introduce the application of protoplast transient assay to compare the efficiency of different CRISPR constructs. The process of protoplast isolation and transformation, and the assessment of genome-editing results, will be covered. The processes of plant regeneration (i.e., the tissue culture process) and transformation are the major hurdles of developing CRISPR in any plant system. We will cover the typical process of tissue culture and possible modifications to improve success. The effects of the type of plant hormone, concentration, combination, and the treatment time on shoot and root regeneration will be discussed. In addition, Agrobacterium-mediated plant transformation and the particle bombardment approach will be introduced and compared. We believe the development of the CRISPR technology in diverse plant systems will broadly facilitate research and will be of great utility to the plant science community. This workshop will complement the Symposium entitled, “Applications of CRISPR Technology Across the Plant Tree of Life,” which will be held later in the conference.

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1 - Florida Museum Of Natural History, 2360 SW ARCHER RD APT 104, GAINESVILLE, FL, 32608, United States
2 - University of Missouri, Division of Plant Sciences, Donald Danforth Plant Science Center, St. Louis, MO
3 - University of Florida, Florida Museum of Natural History, Gainsville, FL
4 - University Of Florida, Florida Museum Of Natural History, PO Box 117800, Gainesville, FL, 32611, United States
5 - University of Florida, Genetics Institute, FL

none specified

Presentation Type: Workshop
Session: W02, Developing CRISPR systems in non-genetic plant models: from plasmid design to transgenic plant
Location: Virtual/Virtual
Date: Sunday, July 18th, 2021
Time: 9:00 AM(EDT)
Number: W02001
Abstract ID:152
Candidate for Awards:None

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