enChIP systems using different CRISPR orthologues and epitope tags

BMC Research Notes
Toshitsugu FujitaHodaka Fujii

Abstract

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. 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 thi...Continue Reading

References

Jan 1, 1987·Molecular and Cellular Biology·R B DuBridgeM P Calos
Aug 4, 2005·Proceedings of the National Academy of Sciences of the United States of America·Hanako IkedaYoshiki Sasai
Oct 1, 2011·Science·Adam J Bogdanove, Daniel F Voytas
Sep 4, 2014·Genes & Development·Melissa M HarrisonJill Wildonger
Nov 3, 2015·Nature Biotechnology·Benjamin P KleinstiverJ Keith Joung
Feb 6, 2016·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Toshitsugu FujitaHodaka Fujii
Jul 29, 2016·Scientific Reports·Toshitsugu FujitaHodaka Fujii
May 6, 2017·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·Toshitsugu FujitaHodaka Fujii
Jun 7, 2017·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Toshitsugu FujitaHodaka Fujii

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Citations

May 2, 2019·BioTechniques·Claude Van CampenhoutVéronique Kruys
May 13, 2020·Biology Methods and Protocols·Toshitsugu Fujita, Hodaka Fujii
Jan 26, 2020·Nature Communications·Marco D CarpenterElizabeth A Heller
Apr 21, 2021·The CRISPR Journal·Hirotaka FujitaHodaka Fujii

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Methods Mentioned

BETA
immunoprecipitation
affinity purification
transfection
electrophoresis
PCR
affinity-purification

Software Mentioned

enChIP

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