GRASP (Genomic Region Affinity Sequestration by CRISPR purification): A plant transformation-independent CRISPR–Cas9 system for affinity purification of specific chromatin loci in isolated plant nuclei

Abstract

DNA–protein interactions play a fundamental role in essential cellular processes such as transcriptional regulation, DNA replication,recombination, and repair. Investigating these interactions is therefore crucial for understanding gene regulatory networks. Existing methodologies can broadly be classified into two conceptual frameworks: protein-centered approaches, which identify genomic regionsbound by specific transcription factors (TFs), and gene-centered approaches, which aim to identify the proteins associated with specific DNAloci. In grapevine (Vitis vinifera), we first exploited previously generated DAP-seq datasets covering 197 transcription factors, mainlyfrom the MYB and WRKY families, to develop an in silico platform for exploring TF–DNAinteractions. This framework enables both protein-centered analyses, allowing visualization of genomic targets for individual TFs, and gene-centered queries, where a selected genecan be interrogated to identify candidate TFs potentially binding its regulatory regions. While this resource is necessarily limited to the transcription factors included in the dataset, it provides a practical example of how gene-centered interrogation of regulatory interactions can be implemented computationally. Independently from this computational framework, we developed a gene-centeredexperimental strategy based on CRISPR/Cas-mediated chromatin targeting, designed to enable the in situ purification of specific genomic regions. This approach aims to capture chromatin fragments associated with defined loci and ultimately identify proteins and potential long-range DNAinteractions linked to these regions. The method was initially validated using telomeric repeats, whichrepresent highly repetitive genomic targets, and subsequently extended to more complex loci including members of the stilbene synthase (STS) gene family, as well as single-copy genes such as Stay-Green and actin. At the current stage, the method allows efficientenrichment and recovery of DNAassociated with the targeted chromatin regions for all tested loci. Proteomic characterization of associated proteins has so far been successfully performed only for telomeric regions, while optimization of protein recovery for less repetitive targetsis currently ongoing. Together, these approaches highlight the potential of combining computational resources and CRISPR-based chromatin targeting strategies to move from protein-centered analyses toward truly gene-centered investigations of regulatory interactions in plant genomes.

Acknowledgements

PIANO NAZIONALE DI RIPRESAE RESILIENZA(PNRR) Missione 4 “Istruzione e Ricerca” – Componente C2 Investimento 1.1,“Fondo per il Programma Nazionale di Ricerca e Progetti di Rilevante Interesse Nazionale (PRIN)”