EVALUATION OF GENE EDITING APPROACHES IN HIGHER PLANTS

Fabiola Guadalupe Ramirez Torres

doi:10.7273/000006530

Advances in gene editing have facilitated new approaches to improve traits of interest in plants. Specifically, CRISPR-Cas9 and associated endonuclease systems allow for the targeting of agriculturally important genes such as disease resistance, improving yield and shelf life, and changing specific traits related to a final high-quality product. SpCas9 uses a tracrRNA: crRNA (gRNA) sequence to target the DNA and induce double-stranded breaks at targeted locations. SpCas9 is widely used for genome editing in mammals and plants; however, other promising endonucleases have been discovered with the potential for greater editing efficiency. Particularly, the nucleases Cas12a (also known as Cpf1) and Cms1 are promising for plant transformation and genome editing approaches. Cas12a, discovered in the bacteria Francisella novicida U112, and Cms1, discovered in the genera Microgenomates and Smithella, are smaller enzymes than SpCas9. Both utilize only a gRNA formed by a mature crRNA to target a T-rich PAM sequence and generate cohesive DNA ends. Although successful gene editing has been achieved in several plants, a single approach that is useful, economically efficient, and reliable to use across different crops has been challenging to develop. The first aim of this work was to answer this question among our candidates: Cas12a, Cms1, and SpCas9. We explore efficiency by targeting the GFP gene in N. benthamiana protoplast as an evaluation system. The generation of gene-edited plants requires identifying the most suitable method to deliver the CRISPR system into the plant cell. Therefore, recalcitrant crops to transformation is a challenge for gene editing in certain crops. Moreover obtaining lines without transgenic elements relies on outcrossing the transformed-edited obtained plant to non-transgenic plants. This outcrossing approach is incompatible with several high-value, obligated outcrossing crops. The second aim of this work is to present a novel method of delivering gene editing reagents across graft junctions using transgenic tobacco as the donor and wild-type tobacco as a scion. This innovation expands the capabilities of plant gene editing to generate non-transgenic edited plants. Implementing gene editing in agriculturally relevant cases is a promising tool for addressing challenges (such as generating climate-resilient crops and creating the next generation of elite cultivars). Grapes were reported to produce 74 million tonnes in 2021 (FAO); 70% of the grape production is used for the wine industry. A promising target for editing in wine grapes, an economically significant crop, is the o-methyltransferase-encoding gene VvOMT3. The VvOMT3 protein is involved in the synthesis of methoxypyrazine, which imparts an undesirable green pepper flavor in wines and produces low anthocyanin content, weak color, and poor tannin quality. Removal of this compound could result in important improvements to flavor. Therefore, the third aim of this work is to target the OMT3 gene, using gene editing, for expression knock-out in organogenic cultures from Vitis vinifera (cv. ‘Cabernet Sauvignon’). Knowledge of the efficacy of multiple nuclease systems, plant tissues in which they are most efficient, and their utility in both fundamental and applied systems, is expected to greatly improve the success of future gene editing projects. As new editing technologies improve and broaden to employ additional endonucleases, the success of inducing changes in genes of interest, such as the proposed OMT modification in grapes, becomes increasingly feasible.

EVALUATION OF GENE EDITING APPROACHES IN HIGHER PLANTS

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