enChIP systems using different CRISPR orthologues and epitope tags

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 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. Results 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 this system as well. Our findings indicate that these tools will increase the flexibility of enChIP analysis.


Introduction
Identification of the molecules associated with a genomic region of interest in vivo is an essential step in understanding the regulatory mechanisms underlying its functions. To this end, we previously developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate genomic regions of interest while retaining their molecular interactions [1,2]. In enChIP, engineered DNA-binding molecules such as transcription activator-like (TAL) proteins [3] or the clustered regularly interspaced short palindromic repeats (CRISPR) system [4,5], which consists of a catalytically inactive form of Cas9 (dCas9) and guide RNA (gRNA), are used to tag the locus of interest, followed by affinity purification of the tagged locus to allow identification of associated molecules. For specific and efficient affinity purification, we usually use 3xFLAG-tag in conjunction with antibody (Ab) against the epitope tag. Locus-tagging can be done in the cell by expressing engineered DNA-binding molecules [1,2,[6][7][8][9] or in vitro using recombinant or synthetic engineered DNAbinding molecules [10,11]. Combination of enChIP with mass spectrometry (MS), RNA sequencing, and nextgeneration sequencing (NGS) has enabled us to identify proteins [1,2,6], RNAs [7], and genomic regions [9,11] interacting with specific genomic regions of interest in a non-biased manner.
In our previous enChIP studies, we used the CRISPR system from Streptococcus pyogenes (S. pyogenes), the most extensively analyzed version of this system. However, because of its requirement for the protospacer adjacent motif (PAM) sequence (5′-NGG-3′), some DNA sequences cannot be targeted by this CRISPR system.
In addition to the S. pyogenes CRISPR system, those from other species have been used for genome editing or

BMC Research Notes
*Correspondence: hodaka@hirosaki-u.ac.jp 1 Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan Full list of author information is available at the end of the article other purposes. Among others, the CRISPR system from Staphylococcus aureus (S. aureus) [12] has been used widely. Since the size of its Cas9 is smaller than that of the S. pyogenes Cas9, the length of its expression plasmid can be shorter than that of the S. pyogenes Cas9, enabling higher efficiency of transfection or transduction. In addition, since its PAM sequence (5′-NNGRRT-3′ or 5′-NNGRR(N)-3′) is distinct from the S. pyogenes system, DNA sequences difficult to be targeted by the S. pyogenes system can be targeted by the S. aureus system, increasing the flexibility of the enChIP technology.
To solve the potential problem of the S. pyogenes CRISPR system and increase the flexibility of enChIP analysis, we developed an enChIP system utilizing the 3xFLAG-tagged CRISPR system from S. aureus. In addition, we developed another enChIP system using the S. pyogenes CRISPR system fused to a different epitope tag. In combination, these tools might be used to constitute a sequential enChIP system with reduced background noise.

Cell lines
The 293T cell line was derived from the 293 cell line isolated human embryonic kidneys (HEK) and transformed with the SV40 large T antigen [13]. The HT1080 cell line was derived from human fibrosarcoma [14] and purchased from ATCC (CCL-121). The 293T cell line, HT1080 cell line, and HT1080 derivatives were maintained in DMEM (Wako) supplemented with 10% fetal calf serum (FCS).

Immunoblot analysis
Nuclear extracts (NE) were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). NE (10 µg) was subjected to immunoblot analysis with anti-FLAG M2 Ab (F1804, Sigma-Aldrich) or Ab against AM-tag (39715, Active Motif ) as described previously [1].

Generation of a lentiviral expression system for enChIP using S. pyogenes CRISPR and 2xAM-tag
The orthologues of Cas9 can be used for sequential enChIP, which might be useful for decreasing the background noise of either system alone. However, for sequential enChIP, CRISPR complexes of different species must be pulled down using different affinity-purification systems (e.g., purification using Abs against each dCas9 or different epitope tags). To this end, we generated a lentiviral expression system of S. pyogenes CRISPR in which Sp-dCas9 was fused with an NLS and the 2xAM-tag (Sp-dCas9-2xAM) (Fig. 2a). The lentiviral expression system allows quick establishment of stable transformants expressing the CRISPR complex even when the target cells are not proliferating. The coding region of Sp-dCas9-2xAM was inserted into the CSII-U6-gRNA-CBh-3xFLAG-PA-dCas9-P2A-Puro plasmid to generate pLenti_dCas9-2xAM. The gRNA dsDNA targeting the IRF-1 promoter was inserted into pLenti_ dCas9-2xAM to generate pLenti_dCas9-2xAM_hIRF-1.
To confirm that the system (Fig. 2b) works, pLenti_ dCas9-2xAM or pLenti_dCas9-2xAM_hIRF-1 was transduced into a human fibrosarcoma cell line, HT1080. After puromycin selection, expression of Sp-dCas9-2xAM was confirmed by immunoblot analysis with Ab against the AM-tag (Fig. 2c).  Next, we examined yields of enChIP for the target IRF-1 promoter region. Cells expressing Sp-dCas9-2xAM in the absence or presence of gRNA targeting the IRF-1 promoter were crosslinked with formaldehyde. The crosslinked chromatin was fragmented by sonication, and complexes containing the CRISPR complexes were subjected to affinity purification using Ab against the AM-tag. The yields of enChIP in the presence of the gRNA were comparable with those obtained using HT1080 expressing 3xFLAG-dCas9 and gRNA targeting the IRF-1 promoter region [16] (Fig. 2d). These results showed that enChIP using the 2xAM-tag can also specifically and efficiently isolate target genomic regions. Because lentivirus can infect non-dividing cells, this enChIP system would also be useful for cells that are not proliferating.
We have not generated stable cell lines expressing Sa-dCas9-3xFLAG. Therefore, at this stage, we cannot compare the enChIP efficiencies between Sa-dCas9-3xFLAG and the lentiviral system of Sp-dCas9-2xAM. However, considering that the enChIP efficiencies between 3xFLAG-Sp-dCas9 and Sa-dCas9-3xFLAG using transient expression systems are comparable (Fig. 1e), and the enChIP efficiencies between 3xFLAG-Sp-dCas9 and Sp-dCas9-2xAM using stable transformants are also comparable (Fig. 2d), we speculate that the enChIP efficiencies between Sa-dCas9-3xFLAG and Sp-dCas9-2xAM might also be comparable.

Conclusions
In this study, we developed an enChIP system using S. aureus CRISPR. Due to its distinct PAM sequence (5′-NNGRRT-3′ or 5′-NNGRR(N)-3′), this system will increase the flexibility of the enChIP technology by increasing the proportion of genomic regions that can be targeted. In addition, we developed another enChIP system using S. pyogenes CRISPR fused with a different epitope tag different than the one used for S. aureus CRISPR. Both enChIP systems achieved high yields of the target locus, and it is expected that their yields would be comparable.

Limitations
Further studies might be necessary to assess the utility of the S. aureus system in enChIP combined with MS and NGS in order to allow comparisons with features of the S. pyogenes system such as background signals, offtarget binding, etc. In addition, it would be of interest to determine whether sequential enChIP analysis could be performed using the S. pyogenes and S. aureus CRISPR systems.

Authors' contributions
HF conceived the idea of potential use of Sa-dCas9 for enChIP and constructed expression plasmids. TF and HF designed and performed experiments and wrote the manuscript. MY performed experiments. HF directed and supervised the study. All authors read and approved the final manuscript.
Author details 1 Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan. 2 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. Fig. 2 enChIP using the S. pyogenes CRISPR system tagged with the 2xAM-tag. a The S. pyogenes CRISPR system tagged with 2xAM-tag for enChIP. The system is composed of a fusion protein Sp-dCas9-2xAM (consisting of Sp-dCas9, an NLS, and a 2xAM-tag) and a gRNA. b Scheme of the enChIP system using S. pyogenes CRISPR tagged with the 2xAM-tag. After expression of Sp-dCas9-2xAM and a gRNA for locus-tagging in target cells, enChIP is performed using an Ab against the AM-tag, as shown in Fig. 1b. c Expression of Sp-dCas9-2xAM. pLenti_dCas9-2xAM or pLenti_dCas9-2xAM_ hIRF-1 was transduced into HT1080 cells. After puromycin selection, expression of Sp-dCas9-2xAM was detected by immunoblot analysis with Ab against AM-tag. d Isolation of the IRF-1 locus by the S. pyogenes CRISPR system tagged with 2xAM-tag. Real-time PCR analysis was performed on chromatin complexes isolated by enChIP. An irrelevant locus (SOX2) was analysed as a negative control. The S. pyogenes CRISPR system tagged with the 3xFLAG-tag was used as a positive control for enChIP (See figure on previous page.)