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…

Influenza di alcuni aspetti ambientali sul contenuto di stilbeni nel vino nell’area della DOC “Sangiovese di Romagna” (Italia)

The ambition of the zonation of the Doc “Sangiovese di Romagna” is described as 25 siti sperimentali, aventi diversa origine geologica, in cui è stato individuato un vigneto omogeneo per la determinazione dei principali parametri viticoli ed enologici. In seguito è stato analizzato il contentto di stilbeni nei vini al fine di indepth il legame con le charatteristiche geopedologiche. The studio describes the positive relationship between the altitude and the content of the trans -piceide nelle province di Forlì and Ravenna and of the trans -resveratrolo a Ravenna. I suoli con maggiore calcare attivo hanno fornito vini più ricchi in stilbeni.

Read More

Characterized one of the largest collections of grapevine rootstocks (non-vinifera)

Microsatellite markers are a valuable tool to facilitate the management of germplasm collections and assess genetic diversity. This study reports the genetic characterization of a large collection of 379 rootstocks and other non-viniferaaccessions maintained at the University of Milan, Italy.

Read More

Chemical characterization of distinctive aroma profiles of Valpolicella and Amarone wines

Valpolicella is an Italian wine producing region, famous for the production of high-quality red wines. A distinctive characteristic of this region is the extensive use of post-harvest withering.

Read More

Moderate wine consumption – part of a balanced diet or a health risk?

Consumption of wine/alcoholic beverages remains a topic of great uncertainty and controversy worldwide. The term “no safe level” dominates the media communication and policy ever since population studies in 2018 [1,2] were published, which denied the existence of a J-curve and suggested that ANY consumption of an alcoholic beverage is harmful to health. The scientific evidence accumulated during the past decades about the health benefits of moderate wine consumption, were questioned and drinking guidelines considered to be too loose.

Read More

Using 1H-NMR combined with chemometrics to discriminate the effect of different cuts and toasting of woods used for grape pomace distillate ageing

The purpose of this research study is to consider new solutions for distillate ageing, in alternative to conventional oak chips or barrels in particular sliced wood and peeled wood were compared to oak cubes, normally employed during both wine and distillate ageing.

Read More