A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells
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 s...
| Main Authors: | , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Elsevier BV
2020
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| Online Access: | https://hdl.handle.net/1721.1/123695 |
| _version_ | 1811075948820299776 |
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| author | Spille, Jan Hendrik Hecht, Micca Grube, Valentin Cho, Won-ki Lee, Choongman Cissé, Ibrahim I. |
| author2 | Massachusetts Institute of Technology. Department of Physics |
| author_facet | Massachusetts Institute of Technology. Department of Physics Spille, Jan Hendrik Hecht, Micca Grube, Valentin Cho, Won-ki Lee, Choongman Cissé, Ibrahim I. |
| author_sort | Spille, Jan Hendrik |
| collection | MIT |
| description | 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. Keywords: CRISPR; Stem cell; MS2; Single RNA imaging; Quantitative microscopy; Live cell imaging |
| first_indexed | 2024-09-23T10:13:32Z |
| format | Article |
| id | mit-1721.1/123695 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T10:13:32Z |
| publishDate | 2020 |
| publisher | Elsevier BV |
| record_format | dspace |
| spelling | mit-1721.1/1236952022-09-30T19:46:11Z A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells Spille, Jan Hendrik Hecht, Micca Grube, Valentin Cho, Won-ki Lee, Choongman Cissé, Ibrahim I. Massachusetts Institute of Technology. Department of Physics Massachusetts Institute of Technology. Materials Research Laboratory 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. Keywords: CRISPR; Stem cell; MS2; Single RNA imaging; Quantitative microscopy; Live cell imaging 2020-02-05T20:07:37Z 2020-02-05T20:07:37Z 2019 2018 2020-01-21T15:19:26Z Article http://purl.org/eprint/type/JournalArticle 1046-2023 https://hdl.handle.net/1721.1/123695 Spille, Jan-Hendrik et al. "A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells." Methods 153 (January 2019): 35-45 © 2018 Elsevier Inc en http://dx.doi.org/10.1016/j.ymeth.2018.09.004 Methods Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier BV Prof. Spille via Barbara Williams |
| spellingShingle | Spille, Jan Hendrik Hecht, Micca Grube, Valentin Cho, Won-ki Lee, Choongman Cissé, Ibrahim I. A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells |
| title | A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells |
| title_full | A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells |
| title_fullStr | A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells |
| title_full_unstemmed | A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells |
| title_short | A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells |
| title_sort | crispr cas9 platform for ms2 labelling of single mrna in live stem cells |
| url | https://hdl.handle.net/1721.1/123695 |
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