Meganuclease-Based Artificial Transcription Factors

Area of Science:

• Synthetic Biology
• Molecular Biology
• Mammalian Cell Engineering

Background:

• Cell engineering aims to embed artificial gene networks in mammalian cells.
• Current artificial transcription factors have limited applications due to orthogonality constraints.

Purpose of the Study:

• To develop a scalable pipeline for producing artificial transcription factors.
• To utilize engineered homing endonucleases (meganucleases) as DNA-binding domains.
• To demonstrate their utility in constructing synthetic gene circuits.

Main Methods:

• Engineered homing endonucleases (meganucleases) with mutations to inactivate nuclease activity.
• Fusion of inactivated meganucleases to transcriptional activator or repressor domains.
• Integration of these constructs into the synNotch receptor system.

Main Results:

• Inactivated meganucleases function as specific DNA-binding domains.
• Fusion proteins effectively induced or suppressed reporter gene expression.
• Demonstrated successful embedding in synNotch for synthetic circuit construction.

Conclusions:

• Inactivated meganucleases are effective DNA-binding domains for synthetic transcription factors.
• This approach offers a scalable method for creating gene regulatory tools in mammalian cells.
• Enables construction of more complex synthetic biology systems.