Silvia Farinati

Chromatin organization regulates genome stability and gene expression by controlling DNA accessibility to transcription factors and regulatory complexes. DNA–protein interactions are commonly studied using chromatin immunoprecipitation (ChIP), which relies on specific antibodies and technically demanding workflows. CRISPR-Cas technologies enable sequence-specific targeting of genomic loci using catalytically inactive Cas9 (dCas9), but most CRISPR-based chromatin capture approaches in plants require transient or stable transformation, limiting their applicability across species, tissues, and conditions. Here we present GRASP (Genomic Region Affinity Sequestration by CRISPR-Purification), a transformation-independent strategy for sequence-specific chromatin isolation directly from purified plant nuclei. In GRASP, dCas9–guide RNA ribonucleoprotein complexes are assembled in vitro and used to capture predefined genomic regions from sonicated chromatin, bypassing transgene expression. Using grapevine (Vitis vinifera) and tomato (Solanum lycopersicum) as model systems, we validated dCas9 binding to telomeric repeats by in vitro assays and CRISPR-FISH. The affinity purification workflow was applied to telomeric, low-copy, and single-copy loci, with enrichment assessed by qPCR and next-generation sequencing. Beyond DNA isolation, GRASP provides a versatile platform for downstream analysis of locus-associated chromatin components, including protein complexes, distal DNA interactions, and chromatin-associated RNAs.

Chromatin organization regulates genome stability and gene expression by controlling DNA accessibility to transcription factors and regulatory complexes. DNA-protein interactions are commonly investigated using chromatin immunoprecipitation (ChIP), which relies on specific antibodies often involving technically demanding protocols. CRISPR-Cas technologies have enabled sequence-specific targeting of genomic loci using catalytically inactive Cas9 (dCas9), but most CRISPR-based chromatin capture approaches in plants require transient or stable transformation to express the CRISPR machinery, limiting their applicability across species, tissues and physiological contexts. Here, we present GRASP (Genomic Region Affinity Sequestration by CRISPR-Purification), a transformation-independent strategy for sequence-specific chromatin isolation operating directly on purified plant nuclei. In GRASP, dCas9-gRNA ribonucleoprotein complexes are used to capture predefined genomic regions from chromatin under native conditions, bypassing the need for transgene expression. Using grapevine and tomato as model systems, we demonstrate efficient and highly specific enrichment of target loci, including telomeric repeats as well as low-copy and single-copy genomic regions, with qPCR and NGS validation. These results establish GRASP as a robust and broadly applicable platform for locus-specific chromatin isolation in plants. Beyond sequence-specific DNA isolation, GRASP establishes a versatile platform for potential downstream analyses of locus-associated chromatin components, including protein complexes, distal DNA-DNA interactions and chromatin-associated RNAs, providing new opportunities to investigate regulatory architecture in plant genomes. GRAPHICAL

Molecular genotyping is a key factor for plant breeding programming and plant variety protection (PVP). However, its potential still remains to be elucidated when considering ornamental plants like Petunia × hybrida. In this study, a petunia breeding clone collection, including sister line groups, was genotyped through double digest Restriction-site Associated DNA sequencing (ddRADseq), and its genetic diversity and structure were studied. In addition to estimating the high genetic similarity observed among sister lines, this approach allowed the unique discrimination of each clone too. Molecular results agreed with genealogy data, supporting the assessment of genotyping effectiveness. In addition, the minimal number of variants able to uniquely discriminate and/or correctly cluster the experimental lines was investigated. The loci number could be reduced to eight to achieve line discrimination, and a method to identify the specific variant sets is presented. Conversely, to preserve the original clustering with minor adjustments, one hundred loci were required and were obtained through minor allele frequency (MAF) filtering. Moreover, analysis of the chromosomal distribution of variants revealed a predominant accumulation in distal regions. Genetic analyses were repeated considering only variants located in coding sequences and results were in agreement with what previously observed, disclosing the potential of the expressed regions for genotyping purposes. Eventually, the applied approach enabled the investigation of SNPs within genes putatively involved in traits of interest. Our findings encourage the adoption of high-throughput and cost-effective sequencing techniques for petunia genotyping aimed at achieving PVP, supporting new variety registration, and developing marker-assisted breeding (MAB) and marker-assisted selection (MAS) strategies.

Molecular genotyping is a key factor for plant breeding programming and Plant Variety Protection. However, its potential still remains to be elucidated when considering ornamental plants like Petunia × hybrida. In this study, a Petunia breeding clone collection, including sister line groups, was genotyped through double digest Restriction-site Associated DNA sequencing (ddRADseq), and its genetic diversity and structure were studied. In addition to estimate the high genetic similarity observed among sister lines, this approach allowed the unique discrimination of each clone too. Molecular results agreed with genealogy data, assessing genotyping effectiveness. In addition, the minimal number of variants able to correctly genotype the population was investigated. The variant number could be reduced to a hundred with minor adjustments in genotyping results. Moreover, analysis of the chromosomal distribution of variants revealed a predominant accumulation in distal regions. Eventually, genetic analyses were repeated considering only variants located in coding sequences and results were in agreement with what previously observed, disclosing the potential of the expressed regions for genotyping purposes. Our findings encourage the adoption of high-throughput and cost-effective sequencing techniques for plant genotyping, to enhance the technological advancement of breeding programs, in particular for ornamental species.

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.

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.

Crops obtained by genome editing (GE) technologies represent a notable and disruptive shift from genetically modified or transgenic plants, outpacing public perception and regulatory frame-works. Multiple aspects of GE are contributing to the advancement of modern agriculture, in-fluencing the market structure by introducing specific changes within the existing genetic blue-print of an organism. Generally, the GE toolbox includes distinct technologies able to incorporate site-specific and pre-determined genomic modifications, exploiting well-established genetic modification strategies. For these reasons, GE-derived approaches are considered new plant breeding methods, known as new breeding techniques (NBTs). Particularly, CRISPR/Cas-based technologies represent a considerable improvement over prior techniques, being more precise, accurate and straightforward for targeted gene editing, in a reliable and reproducible way, with numerous applications. CRISPR/Cas system exploitation for non-editing purposes has also been widely documented, ranging from cell imaging to expression regulation and DNA assembly. Recently, CRISPR/Cas systems have been employed for target detection and could be used to ef-ficiently identify multiple organic chemicals. High-sensitivity and high-specificity detection are two main points of the CRISPR/Cas technology, without needing complex expertise or technical pipelines. This manuscript aims to provide an overview of the main advances of CRISPR/Cas-based systems into new frontiers of non-editing. The associated implications (e.g., molecular traceability) and their relative impacts are also presented and discussed.

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.

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 its independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. Beyond its editing prowess, the discovery of novel Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Researchers have harnessed CRISPR/Cas for gene regulation, deepening insights into gene expression and epigenetic changes. Precisely targeting DNA or RNA sequences, CRISPR/Cas effectively identifies and counters pathogens. Furthermore, it aids in genome imaging and sequencing, revealing genome spatial organization and chromatin dynamics. The non-editing applications of CRISPR/Cas exhibit 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.