A Tunable Long Duration Pulse Generation Circuit in Mammalian Cells

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Previous pulse generator circuits in various systems were limited to short durations (<1 day).
• Longer-lasting pulsatile gene expression is crucial for studying cellular dynamics and applications.

Purpose of the Study:

• To engineer a feedback-based gene circuit for multiday pulsatile gene expression in mammalian cells.
• To achieve tunable duration and amplitude of gene expression pulses using post-transcriptional logic.

Main Methods:

• Constructed a circuit using the PERSIST platform with entirely post-transcriptional logic.
• Engineered inducer-stabilized CRISPR endoRNases responsive to FDA-approved drugs for external regulation.
• Implemented a two-state cross-repression positive feedback topology for pulse generation.
• Optimized circuit design via chromosomal integration and varying stoichiometries.

Main Results:

• Developed tunable pulse generator circuits with durations ranging from 2 to 6 days.
• Achieved multi-day pulsatile gene expression in response to a single inducer input.
• Demonstrated >20-fold change in small molecule responses using engineered proteins.

Conclusions:

• The developed small molecule-stabilized PERSIST proteins are valuable for post-transcriptional gene circuit development.
• Tunable post-transcriptional pulse generators in mammalian cells can advance the study of gene networks and cell-based therapies.