A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells
Introduction
Imaging of endogenous messenger RNA (mRNA) is a powerful tool to reveal dynamic variation of transcriptional activity directly in living cells [1], [2]. Insertion of an array of target sites for the RNA binding coat protein of phage MS2 (MCP) is the most widely used method to study transcription dynamics in vivo. The MS2 system has been used in model systems ranging from bacteria [3], yeast [4], slime mold [5], and eukaryotic cells [2] to live Drosophila [6], [7], [8], zebrafish [9], and mice [10]. Insertion of cassettes encoding 24 or more repeats of the MS2 sequence combined with quantitative fluorescence microscopy enables imaging of single transcripts [11], [12], analysis of mRNA trafficking [13], subcellular localization of translation [14], [15], [16], [17], [18], [19], as well as observation of nascent transcript synthesis at the transcription site [20], [21].
Insertion of an MS2-cassette for tagging of a specific gene in stem cells was successfully demonstrated using a TALEN-based genome editing approach [22]. A CRISPR-based approach [23] could allow more versatile tagging of arbitrary gene loci. Here, we develop a versatile platform for tagging mRNA with a 24xMS2 cassette directly in live cells. We developed a universal cloning strategy employing reusable DNA fragments and highlight an example of labelling endogenous mRNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells (mESCs). Using quantitative fluorescence microscopy we quantified the number of nascent transcripts produced at the Esrrb gene locus in living stem cells. We show that down-regulation of Esrrb expression in a population of cells upon differentiation towards epiblast-like cells (EpiLC) results from an increasing fraction of cells that completely shut down expression of the gene.
Section snippets
Cell culture
R1 mouse embryonic stem cells were a gift from E. Calo (MIT). Cells were cultured in serum-free 2i media as previously described [24], [25]. Cell culture flasks and dishes as well as imaging dishes were coated with 5 μg/ml PLO (Sigma) in PBS buffer and subsequently 5 μg/ml laminin (VWR) in PBS for at least 5 h each at 37 °C. Cell culture media were exchanged every 24 h.
Stable expression of MCP-SNAP
The bacteriophage MS2 stem loop sequence forms an RNA secondary structure that is uniquely recognized by the RNA-binding
Dual color single molecule RNA-FISH
To further verify insertion of the MS2 cassette in one allele and assess potential effects on transcription we performed dual-color single molecule fluorescence in situ hybridization (smFISH). We designed two probe sets targeting either an intronic region of the gene of interest or the MS2 cassette, respectively. The first set targeting intron 3 of the Esrrb locus were designed using the BioSearch Technologies tool (mask 5, probe length 20, 48 probes labelled with Quasar670). The latter set
Dual color single molecule FISH
We first performed fixed cell dual color single molecule FISH experiments on mouse embryonic stem cells to detect both, an intronic region of the Esrrb transcript highlighting the position of the gene locus, and the MS2 cassette (Fig. 3A). As expected we detect two bright foci in the nucleus of most cells in the intronic FISH channel, corresponding to the two alleles from which the gene is transcribed. One of the transcription sites was usually also clearly identified in the MS2 FISH channel,
Discussion
Quantitative imaging of mRNA with single molecule sensitivity directly in single living cells can yield key insights that may be hidden in population studies. The technical hurdle had historically been the insertion in the gene locus of interest of RNA labelling cassettes like MS2 that enable single molecule in vivo characterization. Here, we present a versatile platform for CRISPR/Cas9-based insertion of a widely used 24xMS2 cassette and use it to study the transcriptional activity of a key
Acknowledgements
We thank E. Calo (MIT) for the wild-type R1 cells, and differentiation protocol and L. D. Lavis (HHMI/Janelia) and J. Grimm (HHMI/Janelia) for gift of the JF646-SNAP ligand. pCRISPaint-HaloTag-PuroR was a gift from Veit Hornung (Addgene plasmid # 80960). pDZ415 (24MS2SL loxP-Kan-loxP) was a gift from Robert Singer & Daniel Zenklusen (Addgene plasmid # 45162). pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988).
Funding
This work was supported by the NIH Director’s New Innovator award #DP2CA195769 (to I.I.C). J.-H.S. is supported by a postdoctoral fellowship from the German Research Foundation (DFG, SP1680/1-1).
Declarations of interest
None.
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2022, Handbook of Epigenetics: The New Molecular and Medical Genetics, Third EditionMS2-TRIBE Evaluates Both Protein-RNA Interactions and Nuclear Organization of Transcription by RNA Editing
2020, iScienceCitation Excerpt :Thus, MS2-TRIBE cannot be applied to patient samples to ascertain important protein RNA contacts related to disease, as can be done with CLIP (Luna et al., 2017). However, the genetic engineering required to perform MS2-TRIBE on cultured cells has been made easier with the widespread adaptation of CRISPR insertions (Spille et al., 2019) and coding sequence tagging where hundreds of lines have been made to express MS2 stem loops (Sheinberger et al., 2017; Wan et al., 2019). In addition, a mouse expressing the β-actin gene homozygously tagged with MS2 loops provides a resource for these studies in vivo (Lionnet et al., 2011) and hence the transcriptional environment around the actin locus in a variety of tissues.
The dynamic lifecycle of mRNA in the nucleus
2019, Current Opinion in Cell BiologyCitation Excerpt :For instance, endogenous gene tagging was achieved by gene knock-ins to produce transgenic mice either with MS2 [13] or PP7 [14] sequences, providing the ability to track mRNAs in living tissues [15,16]. Other approaches for genomic integrations are CD-tagging that provides random tagging of endogenous genes [17], TALEN-based genome editing of MS2 and PP7 sequences [18], and a CRISPR-based knock-in approach [19]. Recently, the detection of endogenous mRNAs with CRISPR–Cas enzymes that bind to RNA rather than DNA, has been demonstrated [20••,21].