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…

Soil and nutritional survey of Greek vineyards from the prefecture of Macedonia, Northern Greece, and from the island of Santorini

Vitis vinifera L. is one of the most important cultures for the soil and
climate conditions of Northern Greece and Santorini. However, very little information is provided with regard to its nutritional requirements and critical levels of nutrient deficiencies and toxicities. The aim of this study was to provide an integrated nutritional survey for the Greek conditions of wine and table varieties.

Read More

How and when potential yield disappeared

Every year, after the inflorescences counting in spring, a certain loss in the number of clusters was observed at the harvest.

Read More

Aceto Balsamico Tradizionale di Modena” PDO authenticity: detection of caramel-containing vinegar by HS-GC-IMS

Balsamic vinegars of Modena (Italy), namely Aceto Balsamico di Modena PGI (AB PGI) and Aceto Balsamico Tradizionale di Modena PDO (ABT PDO) are among the most important geographical indication products for Italy. ABT PDO, despite its very limited production, is recognized as one of the most representative Italian artisan gastronomic products, and it is known and commercialized all around the world. The economic value of ABT PDO (“affinato” and “extra-vecchio” types, depending on the aging), prepared following a traditional way and aged for many years in a set of barrels (transferring a certain amount of vinegar from one cask to another in a decreasing “topping up” procedure) is great, when compared to AB industrially prepared with caramel. AB PGI is certainly the most widespread industrial-type vinegar in the world, deriving from low-temperature condensed grape must (or cooked must) mixed with wine vinegar, obtaining balsamic vinegars with a caramel-like taste. Depending on its economic value, ABT PDO is often object of fraud, requiring to fight counterfeit products and imitations.

Read More

Impact of must oxidation on β-lyase coding genes of Saccharomyces cerevisiae and the release of polyfunctional thiols in Sauvignon blanc

Polyfunctional thiols represent a potent class of aroma compounds that shape the wine aroma due to their low odor detection thresholds (Darriet et al., 1995).

Read More

Comparison of the skin resistance of several grape varieties in relation to their physico-chemical properties

The purpose of this study is to compare the skin resistance (SR) of the grapes with physico-chemical propertiess using a stong dataset and multidimentional statistical analysis .
A recent study has shown the role skin resistance plays against pest invasion but skin resistance could be a useful agronomic parameter, for example in the choice of the type of winemaking, by influencing the quantity of juice during crushing and maceration.

Read More