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Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines
Published on: June 2, 2018
Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas
Fahim Farzadfard1, Samuel D Perli, Timothy K Lu
1Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
ACS Synthetic Biology
|August 28, 2013
Summary
Researchers repurposed the CRISPR/Cas system for precise transcriptional regulation in eukaryotic cells. This novel CRISPR-based gene regulation approach simplifies synthetic biology and gene network analysis.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Gene Regulation
Background:
- Transcriptional regulation is crucial for biological systems and synthetic gene circuits.
- Existing methods for synthetic transcriptional regulation require complex protein engineering.
- Scalable and tunable transcription modulation is needed for biological research and applications.
Purpose of the Study:
- To demonstrate a new method for transcriptional regulation using the CRISPR/Cas system.
- To enable scalable, tunable, and simple modulation of transcription in eukaryotic cells.
- To explore applications in synthetic gene circuit construction and natural gene network analysis.
Main Methods:
- Programmed the CRISPR/Cas system from Streptococcus pyogenes to direct gene activation and repression.
- Engineered guide RNAs (gRNAs) for DNA targeting to eukaryotic promoters.
- Demonstrated CRISPR-based transcription factors (crisprTFs) in yeast and human cells.
- Tuned crisprTF activity by altering gRNA binding sites and layering regulatory modules.
Main Results:
- CRISPR/Cas system successfully directed transcriptional activation and repression.
- crisprTF activity was tunable by modifying gRNA targeting and promoter architecture.
- Layering gRNAs with small molecule-responsive proteins enabled externally controllable regulation.
- Single nucleotide promoter changes created orthogonality for gRNA-guided crisprTFs.
Conclusions:
- CRISPR-based eukaryotic gene regulation offers a simplified and scalable approach.
- This technology facilitates the construction of synthetic gene circuits.
- It provides new avenues for mapping natural gene networks and their phenotypic effects.
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