Related Experiment Video
Updated: Mar 6, 2026
10:46
Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
Published on: October 18, 2022
2.3K
Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies
Emil D Jensen1, Raphael Ferreira2, Tadas Jakočiūnas1
1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark.
Microbial Cell Factories
|March 17, 2017
Summary
This study optimized CRISPR-based dCas9 systems for controlling gene expression in yeast. Researchers identified effective guide RNA designs to reprogram metabolic pathways for isoprenoid and triacylglycerol production.
Area of Science:
- Synthetic biology
- Molecular biology
- Biotechnology
Background:
- Transcriptional reprogramming is crucial for cellular adaptation.
- Synthetic biology tools offer orthogonal control of gene expression.
- Nuclease-deficient Cas9 (dCas9) enables targeted gene activation/repression via RNA-guided positioning.
Purpose of the Study:
- Compare dCas9-mediated transcriptional reprogramming systems.
- Optimize gene expression for biobased production of isoprenoids and triacylglycerols (TAGs) in yeast.
- Identify effective guide RNA (gRNA) structures and promoter combinations.
Main Methods:
- Tested 101 gRNA structures across 14 yeast promoters.
- Utilized reporter assays to evaluate gRNA-promoter performance.
- Applied optimized gRNAs for single and multiplex reprogramming strategies.
Main Results:
- Identified gRNA-promoter combinations that perturb gene expression up to threefold.
- Developed constitutive and inducible multiplex reprogramming strategies.
- Achieved significant changes in isoprenoid production and increased TAG levels.
Conclusions:
- Demonstrated comparable performance of constitutive and inducible dCas9 approaches.
- Identified multiplex gRNA designs for significant metabolic pathway perturbation without genomic editing.
- Highlighted the need for improved gRNA design tools and dCas9 engineering.
Related Concept Videos
02:33Combinatorial Gene Control
9.8K
Combinatorial gene control is the synergistic action of several transcriptional factors to regulate the expression of a single gene. The absence of one or more of these factors may lead to a significant difference in the level of gene expression or repression.
The expression of more than 30,000 genes is controlled by approximately 2000-3000 transcription factors. This is possible because a single transcription factor can recognize more than one regulatory sequence. The specificity in gene...
The expression of more than 30,000 genes is controlled by approximately 2000-3000 transcription factors. This is possible because a single transcription factor can recognize more than one regulatory sequence. The specificity in gene...
9.8K
01:24Methods of Nuclear Reprogramming
2.2K
Nuclear reprogramming is a process of transforming one cell type into an unrelated cell type by epigenetic changes that alter the cell’s original gene expression pattern. Such epigenetic changes force cells to express a different set of genes, which play a significant role in inducing transformation into other cell types. Nuclear reprogramming offers applications in reproductive cloning for livestock propagation and regenerative medicine — developing patient-specific cells for...
2.2K