Genome editing (GE) technologies have the potential to completely transform breeding and biotechnology applied to crop species, contributing to the advancement of modern agriculture and influencing the market structure. To date, the GE-toolboxes include several distinct platforms able to induce site-specific and predetermined genomic modifications, introducing changes within the existing genetic blueprint of an organism. For these reasons, the GE-derived approaches are considered like new plant breeding methods, known also as New Breeding Techniques (NBTs). Particularly, the GE-based on CRISPR/Cas technology represents a considerable improvement forward biotech-related techniques, being highly sensitive, precise/accurate, and straightforward for targeted gene editing in a reliable and reproducible way, with numerous applications in food-related plants. Furthermore, numerous examples of CRISPR/Cas system exploitation for non-editing purposes, ranging from cell imaging to gene expression regulation and DNA assembly, are also increasing, together with recent engagements in target and multiple chemical detection. This manuscript aims, after providing a general overview, to focus attention on the main advances of CRISPR/Cas-based systems into new frontiers of non-editing, presenting and discussing the associated implications and their relative impacts on molecular traceability, an aspect closely related to food safety, which increasingly arouses general interest within public opinion and the scientific community.

DNA-binding proteins play a pivotal role in critical cellular processes such as DNA replication, transcription, recombination, repair, and other essential activities. Consequently, investigating the interactions between DNA and proteins is of paramount importance to gain insights into these fundamental cellular mechanisms. Several methodologies have been devised to uncover DNA-protein interactions, which can be broadly categorized into two approaches. The "protein-centered" approach focuses on identifying the DNA sequences bound by a specific transcription factor or a set of TFs. Techniques falling within this category include chromatin immunoprecipitation, and protein-binding microarrays. The "gene-centered" approach entails using one or more DNA sequences as bait to explore the TFs that bind to these specific DNA elements. Methods belonging to this approach encompass yeast one-hybrid (Y1H), PICh, and Reverse ChIP. Both methodologies offer distinct advantages and face limitations, largely stemming from challenges related to complexity, efficiency, and specificity. With the emergence of next-generation sequencing (NGS) protocols and the CRISPR/Cas system, new avenues for investigating trans-cis interactions in organisms have opened. In our research focusing on grapevines, we discuss advancements in both protein- and gene-centered approaches. Firstly, we present the implementation of a DNA Affinity Purification (DAP-seq) protocol in grapevines to explore the cistrome associated with various TFs from the WRKY and MYB families. Secondly, we address the challenge of developing an innovative gene-centered approach utilizing a CRISPR/Cas system for in-situ purification of regulatory elements. This approach aims not only to identify proteins associated with specific genomic regions but also to elucidate long-range DNA interactions.

The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas's groundbreaking attributes lie in its simplicity, versatility, universality, and independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. It is therefore more and more used for genome modification including the generation of mutants. Beyond such editing scopes, the recent development of novel or modified Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Precisely targeting DNA or RNA sequences, the CRISPR/Cas system has been harnessed in fields as diverse as gene regulation, deepening insights into gene expression, epigenetic changes, genome spatial organization, and chromatin dynamics. Furthermore, it aids in genome imaging and sequencing, as well as effective identification and countering of viral pathogens in plants and animals. All in all, the non-editing aspect of CRISPR/Cas exhibits tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.

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.

PIWI, from the German word Pilzwiderstandsfähig, meaning “fungus-resistant”, refers to grapevine cultivars bred for resistance to fungal pathogens such as Erysiphe necator (the causal agent of powdery mildew) and Plasmopara viticola (the causal agent of downy mildew), two major diseases in viticulture. These varieties are typically developed through traditional breeding, often crossbreeding European Vitis vinifera with American or Asian species that carry natural disease resistance. This study investigates the transcriptional profiles of exocarp tissues in mature berries from four PIWI grapevine varieties compared to their elite parental counterparts using RNA-seq analysis. We performed RNA-seq on four PIWI varieties (two red and two white) and their noble parents to identify differential gene expression patterns. Comprehensive analyses, including Differential Gene Expression (DEGs), Gene Set Enrichment Analysis (GSEA), Weighted Gene Co-expression Network Analysis (WGCNA), and tau analysis, revealed distinct gene clusters and individual genes characterizing the transcriptional landscape of PIWI varieties. Differentially expressed genes indicated significant changes in pathways related to organic acid metabolism and membrane transport, potentially contributing to enhanced resilience. WGCNA and k-means clustering highlighted co-expression modules linked to PIWI genotypes and their unique tolerance profiles. Tau analysis identified genes uniquely expressed in specific genotypes, with several already known for their defense roles. These findings offer insights into the molecular mechanisms underlying grapevine resistance and suggest promising avenues for breeding strategies to enhance disease resistance and overall grape quality in viticulture.