A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells

Area of Science:

• Synthetic Biology
• Molecular Biology
• Protein Engineering

Background:

• Cellular processes are driven by protein interactions.
• Controlling engineered biological systems requires precise protein interactions.
• De novo protein design offers a route to novel interaction modules.

Purpose of the Study:

• To design and characterize a set of orthogonal coiled-coil (CC) peptide heterodimers (NICP set).
• To demonstrate the application of the NICP set for regulating cellular localization and transcriptional processes.
• To develop a versatile toolbox for synthetic biology applications.

Main Methods:

• De novo design and synthesis of orthogonal coiled-coil peptide heterodimers (NICP set).
• Demonstration of multiplex cellular localization using CC pairs.
• Engineering CRISPR-dCas9 transcriptional activators with concatenated CC peptide tags (CCC-tag).
• Testing inducible transcription systems in cell culture and in vivo.

Main Results:

• A tunable set of orthogonal CC heterodimers (NICP set) was successfully designed.
• CC pairs enabled multiplexed control of protein localization within single cells.
• CCC-tag facilitated the construction of potent CRISPR-dCas9 transcriptional activators.
• The NICP set effectively tuned gene transcription strength and amplified inducible responses.

Conclusions:

• The NICP set provides a powerful and minimally disruptive module for synthetic biology.
• These engineered protein interactions offer precise control over diverse cellular functions.
• The NICP set and its applications represent a valuable advancement for engineering biological systems.