A Tightly Regulated and Adjustable CRISPR-dCas9 Based AND Gate in Yeast

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2018 Nov 27

PMID 30476163

Citations 10

Authors

Anja Hofmann

Johannes Falk

Tim Prangemeier

Dominic Happel

Adrian Kober

Andreas Christmann

Heinz Koeppl

Harald Kolmar

Affiliations

Soon will be listed here.

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.

Citing Articles

CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges.

Xia Y, Li Y, Shen W, Yang H, Chen X Int J Mol Sci. 2023; 24(20).

PMID: 37894990 PMC: 10607330. DOI: 10.3390/ijms242015310.

Optimization of CRISPR-Cas system for clinical cancer therapy.

Meng X, Wu T, Lou Q, Niu K, Jiang L, Xiao Q Bioeng Transl Med. 2023; 8(2):e10474.

PMID: 36925702 PMC: 10013785. DOI: 10.1002/btm2.10474.

From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems.

Shaytan A, Novikov R, Vinnikov R, Gribkova A, Glukhov G Front Mol Biosci. 2023; 9:1070526.

PMID: 36589238 PMC: 9795063. DOI: 10.3389/fmolb.2022.1070526.

CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids.

Kim S, Kim H, Woo S, Kim T, Rha E, Kwon K Nucleic Acids Res. 2022; 50(22):13155-13171.

PMID: 36511859 PMC: 9825151. DOI: 10.1093/nar/gkac1104.

A Tunable and Expandable Transactivation System in Probiotic Yeast .

Kwak S, Mahmud B, Dantas G ACS Synth Biol. 2021; 11(1):508-514.

PMID: 34939781 PMC: 9013468. DOI: 10.1021/acssynbio.1c00384.

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