Exploring protein–DNA interactions in grapevine through CRISPR–Cas9–guided chromatin precipitation.

Transcription factors are proteins that modulates the expression of genes by binding to cis-regulatory elements (CREs) that act as regulatory sequences of the latter. Their effect on gene expression can change depending on the tissue, organ, the phenological stage of the plant, or environmental conditions, including the exposure to both biotic and abiotic stress. Amongst the strategies used to study these kinds of DNA-protein interactions are protein-centered methods such as Chromatin Immunoprecipitation sequencing (ChIP-seq) or DNA Affinity Purification sequencing (DAPseq), or gene-centered approaches such as Yeast one hybrid assay (Y1H). Although these methodologies are well-established in plants, they face several limitations. While ChIP-seq and DAP-seq identify genomic targets of predefined factors, they cannot resolve the full set of regulators acting on a specific gene; conversely, yeast one-hybrid approaches are DNA-centered but rely on interactions assessed in a heterologous system and outside the native chromatin and cellular context, which may limit their biological relevance. Therefore, new methods are needed to isolate all the proteins at a specific DNA locus. In this study, we developed and applied a CRISPR-Cas9-based method using a catalytically inactive Cas9 (dCas9) to study protein–DNA interactions in plants. In this approach, the dCas9 is guided to a specific DNA region, allowing the DNA and its attached proteins to be pulled down and analyzed. This strategy allows a clearer view of the local proteome at a specific locus. The method was tested in grapevine (Vitis vinifera cv. Sultana) and tomato (Solanum lycopersicum). Telomeric regions were chosen first targets and used to optimize the method and check that its workability, because they have repeated DNA sequences and well-known proteins. Once we were able to pull down the DNA and detect the expected proteins, the method was applied to promoter regions of stress-responsive genes of the stilbene synthases family (STS), aiming at putative MYB-binding sites, to study transcription factor–mediated expression regulation under UV stress. At the end the single locus gene was selected to be more specific like SGR1 in grapevine and GL2 in tomato.