enChIP systems using different CRISPR orthologues and epitope tags

Abstract

Abstract Objective Previously, we developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology, which isolates specific genomic regions while preserving their molecular interactions. In enChIP, the locus of interest is tagged with engineered DNA-binding molecules such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a catalytically inactive form of Cas9 (dCas9) and guide RNA, followed by affinity purification of the tagged locus to allow identification of associated molecules. In our previous studies, we used a 3xFLAG-tagged CRISPR system from Streptococcus pyogenes (S. pyogenes). In this study, to increase the flexibility of enChIP, we used the CRISPR system from Staphylococcus aureus (S. aureus) along with different epitope tags. Results We generated a plasmid expressing S. aureus dCas9 (Sa-dCas9) fused to a nuclear localization signal (NLS) and a 3xFLAG-tag (Sa-dCas9-3xFLAG). The yields of enChIP using Sa-dCas9-3xFLAG were comparable to those using S. pyogenes dCas9 fused with an NLS and a 3xFLAG-tag (3xFLAG-Sp-dCas9). We also generated another enChIP system using Sp-dCas9 fused with an NLS and a 2xAM-tag (Sp-dCas9-2xAM). We obtained high enChIP yields using this system as well. Our findings indicate that these tools will increase the flexibility of enChIP analysis.

Keywords

• enChIP
• Sa-dCas9
• ChIP
• Chromatin immunoprecipitation
• CRISPR