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A portable Cas6f-based system for multiplex translational repression in bacteria.

Giusi Favoino¹, Denis Pšenka², Lea Frideres¹

¹BRiGHT, Technical University of Denmark, Kongens, Lyngby, Denmark.

Nature Communications

|July 8, 2026

Summary

We developed MORTISE, a novel CRISPR-based system for precise gene repression in bacteria. This tool allows for multiplex gene knockdowns, enhancing metabolic engineering applications.

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Area of Science:

Synthetic Biology
Bacterial Genetics
CRISPR Technology

Background:

Engineered small RNAs (sRNAs) offer programmable gene regulation but face challenges in precision, tunability, and multiplexing.
Existing tools can cause genetic burden, rely on host factors, or lack cross-species applicability.
Precise and tunable gene repression is crucial for advancing synthetic biology and metabolic engineering.

Purpose of the Study:

To introduce MORTISE (Multiplex, ORthogonal Translation Interference SystEm), a compact Cas6f-based platform for programmable translational repression in Gram-negative bacteria.
To demonstrate the system's functionality and robustness in diverse bacterial species.
To showcase its application in metabolic engineering for pathway balancing.

Main Methods:

Development of a Cas6f-based system for programmable translational repression.
Testing the system in Escherichia coli and Pseudomonas putida using chromosomal reporter assays.
Implementing single-promoter multiplexing for simultaneous gene knockdowns.
Utilizing a cloning toolbox for efficient assembly of multiple guides.

Main Results:

MORTISE achieves robust, tunable, and multiplexed gene repression in E. coli and P. putida without host Hfq or RNases.
Repression efficiency is enhanced by matching guide and target transcription and targeting the translation initiation region.
Simultaneous knockdowns of up to nine genes are enabled through single-promoter multiplexing.
The system successfully boosted malonyl-coenzyme A-dependent production in P. putida, aiding pathway balancing.

Conclusions:

MORTISE provides a powerful and versatile platform for precise, multiplexed gene repression in Gram-negative bacteria.
The system overcomes limitations of existing tools, offering improved genetic control for synthetic biology.
MORTISE demonstrates significant potential for applications in metabolic engineering and complex genetic circuit design.