Publications
A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells.
JH Spille, M Hecht, V Grube, W Cho, C Lee, II Cissé. Methods 153, 35-45 (2019).
[Link]
Abstract: The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3′ untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.
We had previously used the MS2-system to study transient Pol II clusters at the beta actin locus. The MS2-cassette had been inserted using the PiggyBac system. To be more efficient and flexible we experimented a lot with various strategies to insert the MS2-cassette using CRISPR. This paper presents the solution that we finally came up with and find most usable. The strategy is as follows:
1. Order homology arms (synthesized dsDNA) with an internal multiple cloning site.
2. Ligate the dsDNA into a plasmid backbone.
3. Digest the MS2-cassette (including selectable marker) from a donor plasmid and ligate it into the MCS inside the homology arms to generate a repair template.
4. Co-transfect the repair template with your sgRNA and find a good clone.
We applied this strategy in a number of cell lines with great success. This paper contains a detailed step-by-step manual for all the cloning steps involved. We also demonstrate hoe to use quantitative imaging on a standard epifluorescence microscope to estimate the number of nascent transcripts at the Esrrb gene locus in live mouse embryonic stem cells (~6 on average). During differentiation towards epiblast-like cells Esrrb gets down-regulated over the course of 24h. We show that this happens in a binary fashing - the average number of transcripts at the gene locus remains the same when it is 'on', but in progressively more cells we cannot find the gene locus. The gene is either still 'on' or completely 'off'.