An enChIP system for the analysis of genome functions in budding yeast

Abstract The identification of molecules associated with a specific genomic region is essential for elucidating the molecular mechanisms underlying genome functions such as transcription. Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) is a technology that enables the purification of specific genomic regions and the subsequent identification of their associated molecules. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, such as variants of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 (dCas9) and a guide RNA. This article describes the generation of a plasmid expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and its successful expression in the budding yeast, Saccharomyces cerevisiae. Furthermore, we showed that this plasmid can be used for enChIP analysis in budding yeast. In addition, the plasmid may also be a useful tool for researchers analyzing genome functions such as transcription and for CRISPR interference experiments in budding yeasts.


Introduction
Identification of the regulatory molecules binding to a genomic region of interest is required to understand the molecular mechanisms underlying the regulation of genome functions, including transcription and epigenetic regulation. To achieve this goal, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology, which enables isolation of a genomic region of interest and identification of its associated molecules [1]. Engineered DNA-binding molecules that can be used to tag a target locus include transcription activator-like proteins [2] and the clustered regularly interspaced short palindromic repeats (CRISPR) system [3][4][5], which consist of a catalytically inactive form of Cas9 (dCas9) and a guide RNA (gRNA) (see our recent reviews [6,7] for comprehensive lists of publications using CRISPR-based systems). These engineered molecules can be expressed in a cell of interest to tag a locus (in-cell enChIP) [1] or incubated with fragmented chromatin in vitro (in vitro enChIP) [8]. In vitro enChIP utilizes a functional CRISPR complex consisting of recombinant dCas9 proteins and synthetic gRNA. The locus tagged with the engineered DNAbinding molecules is purified by affinity purification, and the associated molecules are identified by mass spectrometry (for proteins) [1,9] or next-generation sequencing (for RNA and other genomic regions) [10][11][12][13].
This article describes the generation of a plasmid expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) for use in enChIP analyses of the budding yeast, Saccharomyces cerevisiae. The plasmid will be useful for researchers in analyzing genome functions such as transcription by enChIP and may also be useful for analyses of gene functions through loss-of-function experiments using CRISPR interference (CRISPRi).

Transformation of plasmids into YPH499
Transformation of 3xFLAG-dCas9/pTEF1p-CYC1t into yeast cells (65 ng/transformation) was carried out using a MicroPulser TM electroporator (Bio-Rad, Hercules, CA, USA, Cat# 1652100) according to a protocol for budding yeast provided by the manufacturer. After transformation, cells were plated on SD/-TRP medium and allowed to grow for 3 days at 30 C. Transformation of p426-SNR52p-gRNA.CAN1.Y-SUP4t into the yeast cells possessing 3xFLAG-dCas9/pTEF1p-CYC1t was performed by the same method, and cells were plated on SD/-TRP/-URA medium.

Simple Western TM assay and general immunoblot analysis
Yeast cells were suspended in 100 ml (ca. 2.5 volumes of yeast pellet) of a crushing buffer (50 mM sodium phosphate, pH 7.4, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride) and 40 ml of 0.5 mm glass beads (BioSpec Products, Bartlesville, OK, USA, Cat# 11079105), and were crushed with a Bead-Beater (BioSpec Products). Supernatants were recovered after centrifugation at 12,000 rpm for 15 min. Subsequently, 1.2 mg or 6.25 mg of the extract was subjected to Simple Western TM Assay using the "Wes" fully automated system (ProteinSimple, Bio-Techne, MN, USA) with an anti-FLAG M2 antibody (Ab) (Sigma-Aldrich, St Louis, MO, USA, Cat# F1804, RRID: AB_262044) or sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Brilliant Blue (CBB) staining, respectively. Alternatively, the extract (20 mg) was subjected to a general immunoblot analysis with an anti-FLAG M2 Ab as described previously [1] or SDS-PAGE followed by CBB staining.

Results and discussion
To perform analyses using the dCas9 protein in budding yeast, we constructed the 3xFLAG-dCas9/pTEF1p-CYC1t plasmid, in which the expression of 3xFLAG-Sp-dCas9 is driven by the TEF1 promoter (Fig. 1A). The plasmid contains an autonomous replication origin (CEN/ARS). Transformed yeast cells can be selected in a tryptophan-deficient medium.
The 3xFLAG-dCas9/pTEF1p-CYC1t plasmid was transformed into the S. cerevisiae YPH499 strain, and transformants were selected on SD/-TRP plates. The parental YPH499 strain did not grow on this medium, whereas many colonies were observed for transformants.
Expression of 3xFLAG-Sp-dCas9 was analyzed in 10 randomly selected clones. As shown in Fig. 1B and C, all 10 clones showed comparable levels of 3xFLAG-Sp-dCas9, suggesting that the 3xFLAG-dCas9/pTEF1p-CYC1t plasmid is stably retained in this budding yeast strain. These results showed that the transformation efficiency in budding yeast for successful expression of 3xFLAG-Sp-dCas9 is very high (100%). Figure 1D shows an immunoblot analysis and CBB staining of a representative clone (#1). A closer look at the CBB staining image revealed that an extra band of ca. 160 kDa was visible (Fig. 1D, arrowhead in the right panel), suggesting that the expression level of 3xFLAG-Sp-dCas9 in this strain was relatively high.
After deposition of 3xFLAG-dCas9/pTEF1p-CYC1t at Addgene a few years ago, the coding sequence of 3xFLAG-Sp-dCas9 in this plasmid was successfully used for enChIP and other analyses in SK1-derived budding yeast strains [16]. We also succeeded in the locus-specific isolation of a target region when an enChIP analysis was performed with a gRNA targeting the CAN1 gene expressed by transformation with the p426-SNR52p-gRNA.CAN1.Y-SUP4t plasmid [15] (Fig. 2). It is expected that 3xFLAG-dCas9/pTEF1p-CYC1t could also be used to express 3xFLAG-Sp-dCas9 for other applications such as those employing CRISPRi.

Conclusions
This report describes the generation of 3xFLAG-dCas9/pTEF1p-CYC1t, an expression plasmid harboring 3xFLAG-Sp-dCas9, and its stable retention in a budding yeast strain. Expression of the 3xFLAG-Sp-dCas9 protein was confirmed by Simple Western TM assay, immunoblot analysis, and CBB staining. Furthermore, we showed that this plasmid can be used for enChIP analysis in budding yeast. This plasmid will be useful for enChIP analyses and other applications in budding yeast.

Limitations
This report used only a single budding yeast strain, YPH499. It would also be useful to determine whether the 3xFLAG-Sp-dCas9 protein can be expressed from 3xFLAG-dCas9/pTEF1p-CYC1t in other yeast strains. However, since YPH499 is not exceptional in terms of its regulation of transcriptional and translational machineries and other physiological properties [14], it is reasonable to assume that 3xFLAG-dCas9/pTEF1p-CYC1t could indeed be used successfully in other strains. In addition, we did not examine whether the 3xFLAG-Sp-dCas9 protein is expressed when the plasmid is integrated into the chromosome although we do not see why an integrated form of the plasmid would not support expression of 3xFLAG-Sp-dCas9. In this regard, such use has several merits; for example, it would not be necessary to continuously select plasmid-positive cells with selection media. On the other hand, it would be necessary to select multiple clones to obtain one with adequate levels of expression because plasmids randomly integrate into chromosomes, and gene expression is influenced by the genome environment near the integration sites. Although continuous selection is necessary to maintain yeast clones bearing plasmids, the establishment of plasmid-positive clones is easy, as shown in this manuscript, and the different clones showed comparable expression levels of 3xFLAG-Sp-dCas9. Therefore, such a strategy would be convenient for most, but not all, research applications. Figure 2: Isolation of the CAN1 locus by enChIP. One of the YPH499 clones expressing 3xFLAG-Sp-dCas9 was further transformed with the p426-SNR52p-gRNA.CAN1.Y-SUP4t plasmid expressing gRNA targeting the CAN1 locus. After enChIP, qPCR was performed to analyze the yield of the target locus (CAN1). An irrelevant locus (ADE2) was analyzed as a negative control. Error bars represent the standard deviation [n ¼ 3 (dCas9) or 4 (dCas9 þ gRNA)]. The underline in the gRNA target sequence represents the protospacer adjacent motif (PAM) site.