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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

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Bacterial Expression System with Deep Repression and Activation via CRISPR-Cas9.

Valentin A Manuvera^1,2, Pavel A Bobrovsky^1,2, Daria D Kharlampieva¹

¹Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, Moscow, Russia.

The CRISPR Journal

|July 14, 2025

Summary

This study introduces a CRISPR-Cas9 system for controlling toxic gene expression in E. coli. It enables precise gene editing during cultivation, enhancing recombinant protein production by managing toxic peptide expression.

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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Published on: September 2, 2021

Area of Science:

Molecular Biology
Biotechnology
Microbial Genetics

Background:

Incomplete repression of toxic genes hinders cell growth in recombinant E. coli.
Existing methods struggle with tight control over toxic polypeptide expression.

Purpose of the Study:

To develop a novel CRISPR-Cas9-based system for inducible control of toxic gene expression in E. coli.
To enhance recombinant protein production by precisely managing toxic peptide expression.

Main Methods:

Engineered pET-series plasmids with transcription terminators for abortive transcription.
Utilized CRISPR-Cas9 to excise terminators, restoring functional gene expression.
Tested with antimicrobial peptides and green fluorescent protein in E. coli.

Main Results:

Achieved strong, inducible repression of toxic gene expression.
CRISPR-Cas9 mediated excision of DNA fragments to activate gene expression.
Demonstrated effective control over toxic peptide production.

Conclusions:

Developed a robust CRISPR-Cas9 expression system for tightly regulated toxic gene expression.
This system improves E. coli cultivation and recombinant protein yields.
Offers a versatile tool for biotechnological applications involving toxic proteins.