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- Title
enChIP systems using different CRISPR orthologues and epitope tags.
- Authors
Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka
- 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 <italic>Streptococcus pyogenes</italic> (<italic>S. pyogenes</italic>). In this study, to increase the flexibility of enChIP, we used the CRISPR system from <italic>Staphylococcus aureus</italic> (<italic>S. aureus</italic>) along with different epitope tags. Results: We generated a plasmid expressing <italic>S. aureus</italic> 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 <italic>S. pyogenes</italic> 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.
- Subjects
CRISPRS; IMMUNOPRECIPITATION; CHROMATIN; GENOMICS; STAPHYLOCOCCUS aureus; PLASMIDS
- Publication
BMC Research Notes, 2018, Vol 11, p1
- ISSN
1756-0500
- Publication type
Article
- DOI
10.1186/s13104-018-3262-4