An enChIP system for the analysis of genome functions in budding yeast. Issue 1 (17th October 2022)

• Record Type: Journal Article
• Authors: Fujii, Hodaka; Fujita, Toshitsugu
• Is Part Of: Biology methods & protocols. Volume 7:Issue 1(2022)
• Publication Date: 2022-10-17
• Subjects: enChIP -- dCas9 -- ChIP -- chromatin immunoprecipitation -- CRISPR -- yeast -- Saccharomyces cerevisiae; Biology -- Methodology -- Periodicals; 570.1
• DOI: 10.1093/biomethods/bpac025
• Languages: English
• ISSNs: 2396-8923

Abstract

Abstract: The identification of molecules associated with a specific genomic region is essential for elucidating the molecular mechanisms underlying genome functions such as transcription. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) is a technology that enables the purification of specific genomic regions and the subsequent identification of their associated molecules. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, such as variants of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 (dCas9) and a guide RNA. This article describes the generation of a plasmid expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and its successful expression in the budding yeast, Saccharomyces cerevisiae . Furthermore, we showed that this plasmid can be used for enChIP analysis in budding yeast. In addition, the plasmid may also be a useful tool for researchers analyzing genome functions such as transcription and for CRISPR interference experiments in budding yeasts.