MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

Miyuki Yuno; Shoko Nagata; Toshitsugu Fujita; Hodaka Fujii

doi:10.1093/biomethods/bpab013

MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

Biol Methods Protoc. 2021 Jul 9;6(1):bpab013. doi: 10.1093/biomethods/bpab013. eCollection 2021.

Authors

Miyuki Yuno¹, Shoko Nagata^{1

2}, Toshitsugu Fujita^{1

2}, Hodaka Fujii^{1

2}

Affiliations

¹ Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
² Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan.

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.

Keywords: CRISPR; ChIP; MSCV; chromatin immunoprecipitation; dCas9; enChIP.