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.

Download the abstract

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…

DEVELOPMENT OF DISTILLATION SENSORS FOR SPIRIT BEVERAGES PRODUCTION MONITORING BASED ON IMPEDANCE SPECTROSCOPY MEASUREMENT AND PARTIAL LEAST SQUARES REGRESSION (PLS-R)

During spirit beverages production, the distillate is divided in three parts: the head, the heart, and the tail. Acetaldehyde and ethanol are two key markers which allow the correct separation of distillate. Being toxic, the elimination of the head part, which contains high concentration of acetaldehyde, is crucial to guarantee the consumer’s health and security. Plus, the tail should be separated from the heart based on ethanol concentration.

Read More

LARGE SURVEY OF THE CHEMICAL COMPOSITION OF WINES RESULTING OF THE PRESSING OF RED WINE MARC. FIRST RESULTS

In the Bordeaux vineyards, press red wine represents about 15% of the volume of wines. Valuing this large volume of press wine is necessary from an economic point of view, of course, but also because of their organoleptic contribution to the blend. Nevertheless, there is a lack of recent knowledge on the composition of press wines. This work aims to establish an initial assessment of their composition (aromatic and polyphenolic) and to set up hypothesis on to the links with their sensorial identity.

Read More

Applying value proposition design to collective strategic actions in family wineries: enhancing territorial resources in Vale dos Vinhedos, Brazil

The study aims to propose collective strategic actions for family wineries, promoting their competitiveness and the valorization of territorial resources.

Read More

Effect of irrigation regime on carbon isotope ratio (δ13c) in different grapevines

In Castilla-La Mancha as other winegrowing regions, vineyards suffer the effects of the global climate warming. Severe spring and summer droughts are increasingly frequent, which concur with the phenological stages most susceptible to water and temperature stress. Under these conditions, irrigation use is required in order to ensure the vineyard growing sustainability. However water resources are increasingly limited, for this reason is required to choose cultivars displaying high water use efficiency.

Read More

Targeted and untargeted 1H-NMR analysis for sparkling wine’s authenticity

Studies on wineomics (wine’s metabolome) have increased considerably over the last two decades. Wine results from many environmental, human and biological factors leading to a specific metabolome for each terroir. NMR metabolomics is a particularly effective tool for studying the metabolome since it allows the rapid and simultaneous detection of major compounds from several chemical families.1 Quantitative NMR has already proven its effectiveness in monitoring the authenticity of still wines.

Read More