Hofmann, Anja ; Falk, Johannes ; Prangemeier, Tim ; Happel, Dominic ; Köber, Adrian ; Christmann, Andreas ; Koeppl, Heinz ; Kolmar, Harald (2018):
A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast.
In: Nucleic Acids Research, 47 (1), pp. 509-520. Oxford Academic, ISSN 0305-1048,
DOI: 10.1093/nar/gky1191,
[Article]
Abstract
The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor. We hence developed a predictable and modular, versatile expression control system. The selection of a reporter gene set up combining a gene of interest (GOI) with a fluorophore by the ribosomal skipping T2A sequence allows to adapt the system to any gene of interest without losing reporter function. In order to obtain a better understanding of the underlying principles and the functioning of our system, we backed our experimental findings with the development of a mathematical model and single-cell analysis.
Item Type: | Article |
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Erschienen: | 2018 |
Creators: | Hofmann, Anja ; Falk, Johannes ; Prangemeier, Tim ; Happel, Dominic ; Köber, Adrian ; Christmann, Andreas ; Koeppl, Heinz ; Kolmar, Harald |
Title: | A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast |
Language: | English |
Abstract: | The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor. We hence developed a predictable and modular, versatile expression control system. The selection of a reporter gene set up combining a gene of interest (GOI) with a fluorophore by the ribosomal skipping T2A sequence allows to adapt the system to any gene of interest without losing reporter function. In order to obtain a better understanding of the underlying principles and the functioning of our system, we backed our experimental findings with the development of a mathematical model and single-cell analysis. |
Journal or Publication Title: | Nucleic Acids Research |
Volume of the journal: | 47 |
Issue Number: | 1 |
Publisher: | Oxford Academic |
Divisions: | 18 Department of Electrical Engineering and Information Technology 18 Department of Electrical Engineering and Information Technology > Institute for Telecommunications > Bioinspired Communication Systems 18 Department of Electrical Engineering and Information Technology > Institute for Telecommunications 05 Department of Physics 05 Department of Physics > Institute for condensed matter physics (2021 merged in Institute for Condensed Matter Physics) 05 Department of Physics > Institute for condensed matter physics (2021 merged in Institute for Condensed Matter Physics) > Statistische Physik und komplexe Systeme 07 Department of Chemistry 07 Department of Chemistry > Fachgebiet Biochemie 07 Department of Chemistry > Fachgebiet Biochemie > Allgemeine Biochemie |
Date Deposited: | 03 Feb 2019 20:55 |
DOI: | 10.1093/nar/gky1191 |
URL / URN: | https://doi.org/10.1093/nar/gky1191 |
URN: | urn:nbn:de:tuda-tuprints-84327 |
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