enChIP systems with MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9

Objective: Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specic genomic regions to facilitate identication of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., 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 exibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. Results: 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 it is feasible to purify target genomic regions with high yields using these plasmids. These systems might give enChIP users greater exibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze different types of target cells.


Method Details
To understand the molecular mechanisms underlying regulation of genome functions such as epigenetic regulation and transcription, it is necessary to identify the regulatory molecules binding to a genomic region of interest. We recently developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology for isolation of genomic regions of interest to facilitate identi cation of their associated molecules [1,2]. The engineered DNA-binding molecules that can be used to tag the target locus include transcription activator-like (TAL) proteins [3] and the clustered regularly interspaced short palindromic repeats (CRISPR) system [4,5] consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9) (see our recent review [6] for the CRISPR-based systems). Locus-tagging can be achieved by expression of engineered DNA-binding molecules (in-cell enChIP) [1,2,[7][8][9][10]. Alternatively, it can be achieved in vitro using recombinant or synthetic engineered DNA-binding molecules (in vitro enChIP) [11,12]. After a target locus is tagged using an engineered DNAbinding molecule, it is isolated by a nity puri cation. Subsequently, associated proteins are identi ed by mass spectrometry (MS) [1,2,7], and associated nucleic acids such as RNAs and other genomic regions are identi ed by next-generation sequencing (NGS) [8,10,12].
In this study we generated murine stem cell virus (MSCV)-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 and various drug resistance genes. Using these plasmids, we were able to purify target genomic regions with high yields. These systems might give enChIP users greater exibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze different types of target cells.

Procedures Transduction of retroviral plasmids
To increase the exibility of enChIP and expand the range of target cells, we constructed retroviral plasmids expressing S. pyogenes dCas9 fused with the 3xFLAG-tag (3xFLAG-Sp-dCas9), using the MSCV system along with various drug selection markers.

enChIP analysis
After expansion, 2 × 10 6 cells were subjected to enChIP analysis. Brie y, the cells were crosslinked with formaldehyde, and the crosslinked chromatin was fragmented by sonication. Subsequently, fragmented chromatin tagged with the CRISPR complex was puri ed using anti-FLAG antibody. enChIP-real-time PCR to calculate the yields of the target genomic region was performed as previously described [13]. The primers used in this study were reported previously [1]. As shown in Fig. 2, the yields of enChIP (7 -9% of input) were high and comparable to those obtained by other systems (e.g., ca. 10% of input for the pMXs system [13]). These results revealed that the enChIP system using the MSCV retroviral vectors can be used for downstream applications such as identi cation of molecules associated with the target genomic regions.

Declaration of interests:
The authors declare the following nancial interests/personal relationships which may be considered as potential competing interests: