Implementation of a CRISPR-Based System for Gene Regulation in .

mSphere

Departamento de Microbiología y Parasitología, Facultad de Farmacia, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Universidad Complutense de Madrid, Madrid, Spain

Published: February 2019

AI Article Synopsis

  • CRISPR is not just a gene-editing tool; it can also regulate gene expression by combining Cas9 with transcriptional repressors or activators.
  • Researchers created a system in yeast that shows specific gene repression or activation, demonstrating success through various techniques like qPCR and flow cytometry.
  • The study highlights the potential of CRISPR technology to manipulate complex regulatory traits in important fungal pathogens, enhancing our understanding of their virulence pathways.

Article Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) methodology is not only an efficient tool in gene editing but also an attractive platform to facilitate DNA, RNA, and protein interactions. We describe here the implementation of a CRISPR-based system to regulate expression in the clinically important yeast By fusing an allele of Cas9 devoid of nuclease activity to a transcriptional repressor (Nrg1) or activator (Gal4), we were able to show specific repression or activation of the tester gene , encoding the cytosolic catalase. We generated strains where a 1.6-kbp upstream regulatory region of controls the expression of the green fluorescent protein (GFP) and demonstrated the functionality of the constructs by quantitative PCR (qPCR), flow cytometry, and analysis of sensitivity/resistance to hydrogen peroxide. Activation and repression were strongly dependent on the position of the complex in this regulatory region. We also improved transcriptional activation using an RNA scaffolding strategy to allow interaction of inactive variants of Cas9 (dCas9) with the RNA binding protein MCP (monocyte chemoattractant protein) fused to the VP64 activator. The strategy shown here may facilitate the analysis of complex regulatory traits in this fungal pathogen. CRISPR technology is a new and efficient way to edit genomes, but it is also an appealing way to regulate gene expression. We have implemented CRISPR as a gene expression platform in using fusions between a Cas9 inactive enzyme and specific repressors or activators and demonstrated its functionality. This will allow future manipulation of complex virulence pathways in this important fungal pathogen.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374588PMC
http://dx.doi.org/10.1128/mSphere.00001-19DOI Listing

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