![]() The hybrid of CRISPR-Tag and MS2 loops was directly synthesized by GenScript and then cloned into the intron region of BFP or mCherry, resulted in the final generation of TriTag (BFP-TriTag or mCherry-TriTag). To achieve simultaneous imaging of DNA, nascent RNA, and protein of a specific gene, we designed three versions of TriTags, which are hybrids of fluorescent protein, CRISPR-Tag ( 27) and MS2 loops. To observe transcriptional dynamics of CMV and SFFV promoters, TriTag (BFP) was cloned to the corresponding backbone vectors by T4 DNA ligase. To build the doxycycline-inducible system ( Supplementary Figure S3), mCherry (intron-MS2V5 17X) was cloned to the lentiviral Tet-On 3G inducible vector using T4 DNA ligase. MS2 nx (PCR template: Addgene #27118) ( 18), or MS2V5 nx was then cloned into the BstXI site using T4 DNA ligase. A restriction site BstXI was artificially embedded in the intron region for the ease of molecular cloning. To specifically detect nascent RNAs, the fourth intron of human HSPA5 gene was inserted into mCherry- or BFP-coding sequence. To build plasmids for live-cell RNA labeling ( Supplementary Figure S1), the DNA fragment 12xMS2V5 amplified from Addgene #84561 ( 28) was integrated into the 5′UTR or 3′UTR of mCherry in our lentiviral backbone vector pHR-SFFV-mCherry (same backbone with Addgene #80409) using T4 DNA ligase (New England Biolabs). Fragments of both stdMCP and tdTomato were cloned into a lentiviral vector phage-ubc (Addgene #40649) using NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs). To build stdMCP-tdTomato, the DNA sequence encodes stdMCP was amplified from an Addgene plasmid #104999 ( 26). The following plasmids were specifically constructed for this study. The construction of dCas9-GFP11 14X and GFP1–10 plasmids has been described in our previous study ( 27). The Addgene plasmids #40649 plasmid ( 25) and # 104999 ( 26) were used to express tdMCP-GFP and stdMCP-stdHaloTag, respectively. Cells used in this study were maintained in mycoplasma-free status. All cells were cultured at 37☌ and 5% CO 2 in a humidified incubator. U2OS was cultured in McCoy's 5A (Procell) supplemented with 10% FBS, 1% penicillin and streptomycin. HeLa cells and HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) with high glucose (Gibco) in 10% FBS (Hyclone) and 1% penicillin/streptomycin (Gibco). This tag enables simultaneous real-time imaging of chromatin dynamics (DNA), transcriptional status (RNA) and protein turnover in a single living cell. Here, we report a small tag (∼1.5 kb) which can be genetically inserted into the endogenous locus at the N- or C-terminus of a protein-coding gene by genome editing. However, every copy of the transgene in this system is ∼20 kb in length, which does not aim at endogenous gene tagging. This system is composed of a 200-copy transgene array by combining the lac operator/lac repressor to label DNA ( 31, 32), the MS2-MCP system to tag RNA, and the fluorescent tag to report protein. However, to explore chromatin dynamics and their relation to transcriptional status, it is highly desirable to combine multiple monitoring systems in a single cell.Ī pioneering study created a tetracycline-inducible system to monitor gene expression at the levels of DNA, RNA, and protein in living cells ( 24). Analysis of protein turnover by fluorescent probes was widely performed to elucidate gene expression as well ( 22, 23). The use of the MS2-MCP approach to monitoring multiple steps of the RNA life cycle in real-time has provided unprecedented insights into the process of gene expression ( 18–21). We had previously developed the dCas9-FP system for imaging endogenous genomic DNA, which allows tracking of chromatin dynamics ( 16, 17). Fluorescent tools are leading a revolution in illuminating various cellular molecules, including genomic DNA, RNA and protein ( 9–15). Specific labeling of biomolecules with fluorescent tags in living cells is the cornerstone of cell biology studies. It is becoming increasingly apparent that chromatin remodeling, transcription regulation, and protein expression are interconnected at the cellular level, thus, it is important to understand how they are coordinated in living cells ( 3–8). However, our understanding of how specific genes are spatiotemporally regulated remains incomplete. The establishment of complex transcriptional programs with temporal and spatial precision is crucial for many biological processes ( 1, 2).
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