MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis. Issue 1 (9th July 2021)

• Record Type: Journal Article
• Authors: Yuno, Miyuki; Nagata, Shoko; Fujita, Toshitsugu; Fujii, Hodaka
• Is Part Of: Biology methods & protocols. Volume 6:Issue 1(2021)
• Publication Date: 2021-07-09
• Subjects: enChIP -- dCas9 -- ChIP -- chromatin immunoprecipitation -- CRISPR -- MSCV; Biology -- Methodology -- Periodicals; 570.1
• DOI: 10.1093/biomethods/bpab013
• Languages: English
• ISSNs: 2396-8923

Abstract

Abstract: Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, for example, a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that by using these plasmids, it is feasible to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells.