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A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast

Hofmann, Anja and Falk, Johannes and Prangemeier, Tim and Happel, Dominic and Köber, Adrian and Christmann, Andreas and Koeppl, Heinz and Kolmar, Harald (2018):
A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast.
In: Nucleic Acids Research, Oxford Academic, pp. 509-520, 47, (1), ISSN 0305-1048,
DOI: 10.1093/nar/gky1191,
[Online-Edition: https://doi.org/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
Erschienen: 2018
Creators: Hofmann, Anja and Falk, Johannes and Prangemeier, Tim and Happel, Dominic and Köber, Adrian and Christmann, Andreas and Koeppl, Heinz and 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: 47
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
05 Department of Physics > 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
Official URL: https://doi.org/10.1093/nar/gky1191
URN: urn:nbn:de:tuda-tuprints-84327
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