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

Hofmann, Anja ; Falk, Johannes ; Prangemeier, Tim ; Happel, Dominic ; Köber, Adrian ; Christmann, Andreas ; Koeppl, Heinz ; Kolmar, Harald (2019)
A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast.
In: Nucleic Acids Research, 2018, 47 (1)
doi: 10.1093/nar/gky1191
Article, Secondary publication

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: 2019
Creators: Hofmann, Anja ; Falk, Johannes ; Prangemeier, Tim ; Happel, Dominic ; Köber, Adrian ; Christmann, Andreas ; Koeppl, Heinz ; Kolmar, Harald
Type of entry: Secondary publication
Title: A tightly regulated and adjustable CRISPR-dCas9 based AND gate in yeast
Language: English
Date: 22 November 2019
Place of Publication: Darmstadt
Year of primary publication: 2018
Publisher: Oxford Academic
Journal or Publication Title: Nucleic Acids Research
Volume of the journal: 47
Issue Number: 1
DOI: 10.1093/nar/gky1191
URL / URN: https://doi.org/10.1093/nar/gky1191
Origin: Secondary publication via sponsored Golden Open Access
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.

URN: urn:nbn:de:tuda-tuprints-84327
Classification DDC: 500 Science and mathematics > 530 Physics
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 > Clemens-Schöpf-Institut > Fachgebiet Biochemie
07 Department of Chemistry > Clemens-Schöpf-Institut > Fachgebiet Biochemie > Allgemeine Biochemie
Date Deposited: 03 Feb 2019 20:55
Last Modified: 20 Oct 2023 08:34
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