terclim by ICS banner
IVES 9 IVES Conference Series 9 Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Preliminary steps of a protocol to isolate transcription factors bound to a specific DNA locus in grapevine using CRISPR-dCas9 system

Abstract

Cis-acting regulatory elements are DNA sequences that can be bound by transcription factors to regulate the expression of genes in a condition-dependent and tissue-specific way. It is nowadays possible to search for DNA motives and sequences that a given transcription factor is binding or at least can, but it is still hard to have a glance at all the transcription factors that are contemporaneously located at the same locus. Inspired by an existing technique that uses the CRISPR-Cas system in mammal cells, we are trying to develop a protocol to study such regulation in Vitis vinifera. Using the highly sequence-specific binding capacity of a catalytically inactive Cas9 protein (dCas9), our idea is to set up a system to target a desired sequence and precipitate all the crosslinked proteins and distantly interacting chromatin at this locus and analyze them. After conducting preliminary assays on protoplast system, we got introduced to the CRISPR-FISH technique, that uses dCas9 and a fluorescent guide to label telomeres on nuclei directly isolated from a small quantity of fixed leaves with a very simple procedure. We used part of such technique to easily obtain the chromatin that was needed for our trials and eventually performed the pull-down of the targeted DNA sequences directly on these nuclei after transforming them with the dCas9 complex. Sequencing the obtained fragments allows to verify the specificity of the tool. Far from having eliminated the idea of using protoplasts as model system, we proceed developing both approaches in parallel.

DOI:

Publication date: June 13, 2024

Issue: Open GPB 2024

Type: Poster

Authors

Aurélien Devillars1*, Silvia Farinati1, Edoardo Bertini2, Chiara Fattorini2, Sara Lissandrini2, Adriana Fernanda Soria Garcia1, Bhanu Prakash Potlapalli3, Andreas Houben3, Sara Zenoni2 and Alessandro Vannozzi1

1 Department of Agriculture Food Natural Resources Animals and Environment, University of Padova, Agripolis, Legnaro, Italy
2 Department of Biotechnology, University of Verona, Verona, Italy
3 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany

Contact the author*

Keywords

Molecular Biology, Grapevine, Gene Regulation, CRISPR-Cas9, Protoplasts

Tags

IVES Conference Series | Open GPB | Open GPB 2024

Citation

Related articles…

The chances for using non-saccharomyces wine yeasts for a sustainable winemaking

Climate changes and the trend towards organic and more sustainable winemaking highlighted the need to use biological methodologies. The reduction in the use of SO2, the need of the reduction of ethanol content of wines and the now need to reduce or eliminate chemical phytosanitary products, have prompted the search for alternative practices.

Unravel the underlying mechanisms of delaying ripening techniques in grapevine

In a scenario of changing climate conditions, grapevine is significantly affected at multiple levels. Advancements in phenology and berry ripening, however, are the major dynamics of the generalized increase in average temperature and evaporative demand, negatively affecting berry quality and productivity. The aim of this work was to unravel the underlying mechanisms of bunch-zone auxin application (NAA; 1-Naphthaleneacetic acid) and source-limiting canopy management approaches in delaying berry ripening. In randomized block design experiments, control vines were compared to vines treated with NAA, subjected to apical-to-bunch defoliation or antitranspirant application (n=10-to-42 plants per treatment).

Genetic traceability of the varietal origin of wines: a robust application for must and wines during alcoholic fermentation

Industry and regulatory agencies have developed regulations to ensure authenticity and compliance with wine composition limits. However, this can be truncated by the absence of simple and robust analytical methodologies, uninfluenced by the environment, different oenological techniques and cultural practices. Genetic fingerprinting is the most powerful tool for unequivocal varietal identification; it is not affected by the environment or agronomic practices; however, its usefulness in musts and wines has been controversial and there is currently no routine certification of varietal origin based on DNA analysis.

Polyphenols in kombucha: Metabolomic analysis of biotransformations during fermentation

Kombucha is a non-alcoholic beverage made of sugared tea that is transformed by a symbiotic consortium of yeasts and bacteria. This beverage is increasingly produced at industrial scale, but its quality standards remain to be defined. Metabolomics analysis was carried out using FT-ICR-MS to understand the chemical transformations induced by the production phases and the type of tea on

Modernizing grapevine rootstock breeding at UC Davis: strategies, tools, and early outcomes

Soil salinity is an increasingly critical challenge for grapevine production, particularly in Mediterranean climates like California. Chloride (Cl-) accumulation, in particular, severely impacts vine growth, productivity, and fruit quality.