Eleanor Jane Brant

The 2023 In Vitro Biology Meeting featured an oral presentation competition for Plant Biotechnology Post-Doctoral Associates. Presenters were evaluated on experimental design, data analysis, proper interpretation of the results, originality of the study, technical difficulty, and presentation skills. Our expert panel of judges consisted of Prakash Kumar, National University of Singapore, Deepika Chauhan, Pairwise, Lori Marcum, Corteva Agriscience, and Carlos M. Hernandez-Garcia, CTC Genomics. The judges recognized Isidre Hooghvorst (University of Florida, USA) with the 1st place award for his presentation on Targeted Multi-allelic Epigenetic Editing of Highly Polyploid Sugarcane. Gen Li was (University of Maryland, USA) recognized with 2nd place. Both winners were presented with a certificate and a cash award. We encourage all Plant Biotechnology Post-Doctoral Candidates to consider this as an opportunity to develop their presentation skills at future meetings.

Submitted by Ahmad Omar

First Place

Targeted Multi-allelic Epigenetic Editing of Highly Polyploid Sugarcane

Eleanor Jane Brant

Isidre Hooghvorst

Sugarcane provides 80% of the worlds table sugar and 25% of the global biofuel supply. Genetic improvement of sugarcane is challenging due to its complex, highly polyploid genome (2n = 100-130, x = 10-13). Gene editing with designer nucleases like CRISPR/Cas9 is revolutionizing crop improvement. However, the generation of loss of function mutants is more challenging in polyploid crops with high genetic redundancy. Epigenetic editing could be especially useful for modulating gene expression. For altering the epigenetic state catalytically inactive dCas9, translationally fused with enzymes such as DNA methylases, histone acetyltransferases, or deacetylases, can be targeted to precise genomic locations with the help of sgRNAs. This may exert site-specific control over modifications to DNA, histones, and chromatin architecture for modulation of gene expression. The aim of this study is to explore the targeted epigenetic editing in sugarcane using a dCas9 fusion protein with a DNA methyltransferase targeting the Magnesium Chelatase (MgChl) gene, which is a critical enzyme for chlorophyl biosynthesis. Two epigenetic editing vectors were independently delivered into embryogenic sugarcane callus with biolistic gene transfer targeting different locations of the MgChl gene with 10 replications per recombinant DNA construct. We confirmed the transgenic nature of regenerated plants with PCR, evaluated the greenness of the phenotype using a SPAD-meter and analyzed the expression of MgChl by RT-qPCR. CpG methylation in the MgChl gene was analyzed by Sanger sequencing of cloned PCR amplicons from bisulfite-treated genomic DNA and compared with non-modified sugarcane. Data describing the phenotype of the regenerated plants, corresponding gene expression of the target gene and epigenetic changes of the target gene will be presented. Our findings enable additional research that will explore how epigene editing can be tailored to alter expression of specific target genes in crops.

Isidre Hooghvorst, Agronomy Department, University of Florida-IFAS and DOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville FL 32611. Abstract presentation P-1001.

Second Place

Encapsulation of Long dsRNA in Extracellular Vesicles, SARS-CoV2 Virus-like Particles, and Liposomes for the Stimulation of Antiviral RNA Interference Against Human Coronaviruses

Eleanor Jane Brant

Gen Li

Cas12a (Cpf1), belonging to Class 2, Type V-A CRISPR system, has been applied in a variety of plant species as a promising tool for genome editing. Some specific characteristics made Cas12a distinct from Cas9 and expanded the power of genome-editing tools. Although various engineered Cas12a has been reported to enhance temperature tolerance and relax PAM requirement, the editing efficiency is still relatively low (< 50%) in most plant species. Thus, there is substantial necessity of engineering Cas12a to enhance editing activity in plants. To improve the editing efficiency of LbCas12a, we conducted saturation mutagenesis in E. coli and identified 1977 positive point mutations of LbCas12a. Then, 24 single mutations and various combinational mutations were assessed in human cell, which identified an optimal variant LbCas12a-RVQ. Subsequently, four engineered LbCas12a, Lb-RV, Lb-RRV, Lb-RVQ and Lb-RRVQ were selected to test in different plant species, rice, tomato and poplar. In rice and tomato protoplast, Lb-RV showed more robust genome editing efficiency compared with Lb-WT regardless of temperature conditions. Moreover, Lb-RRV outperformed other three LbCas12a variants at most target sites in rice protoplast via plasmid delivery. In rice T0 plants, Lb-RRV showed high editing efficiency at all TTTV sites. More important, Lb-RRV also enhanced editing efficiency and performed higher biallelic editing (up to 100%) than other LbCas12a variants at TTV target sites that are not preferred PAM for LbCas12a. Moreover, Lb-RRV outperformed other Cas12a variants at all target sites in poplar T0 plants with up to 100% biallelic editing efficiency and especially also enhanced low-activity sites. Taken together, Lb-RRV showed remarkable genome editing efficiency and great potential for precise genome editing and plant breeding.

Gen Li, Department of Plant Science and Landscape Architecture, University of Maryland, College Park, College Park, MD. Abstract presentation P-1003

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