Abstract
Expanding the number of available RNA-binding proteins (RBPs) is vital to establishing posttranscriptional circuits in mammalian cells. We focused on CRISPR-Cas systems and exploited Cas proteins for their versatility as RBPs. The translation of genes encoded in an mRNA becomes regulatable by a Cas protein by inserting a crRNA/sgRNA sequence recognizable by the specific Cas protein into its 5′UTR. These Cas protein-responsive switches vastly expand the available tools in synthetic biology because of the wide range of Cas protein orthologs that can be used as trigger proteins.
Here, we describe the design principle of Cas protein-responsive switches, both plasmid and RNA versions, using Streptococcus pyogenes Cas9 (SpCas9) as an example and show an example of its use in mammalian cells, HEK293FT cells.
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Abbreviations
- crRNA:
-
CRISPR RNA
- mRNA:
-
messenger RNA
- RBPs:
-
RNA-binding proteins
- sgRNA:
-
single guide RNA
- SpCas9:
-
Streptococcus pyogenes Cas9
- tracrRNA:
-
trans-activating crRNA
- UTR:
-
untranslated region
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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Hirosawa, M., Saito, H. (2024). RNA Switches Using Cas Proteins. In: Ceroni, F., Polizzi, K. (eds) Mammalian Synthetic Systems. Methods in Molecular Biology, vol 2774. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3718-0_12
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DOI: https://doi.org/10.1007/978-1-0716-3718-0_12
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