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Updated: Mar 10, 2026

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

Multi-Targeting Non-Specific Genome Engineering in Bacteria.

Runze Sun¹, Ruixiang You^1,2, Yiwen Zhou^1,2

¹State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)

|March 9, 2026

Summary

A new genome engineering method, MNGE, enables stable, multi-copy integration of metabolic genes in diverse bacteria. This approach enhances the production of valuable compounds like fungicides and antibiotics.

Keywords:

bacteria genome engineering microbial drugs multi‐targeting integrases synthetic biology

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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Published on: April 11, 2019

Area of Science:

Microbiology
Synthetic Biology
Metabolic Engineering

Background:

Genome engineering is vital for metabolic engineering and synthetic biology.
Stable gene expression requires host-independent integration technologies for diverse bacteria.

Purpose of the Study:

To develop a generalizable genome engineering approach for stable, multi-copy gene integration in diverse bacteria.
To demonstrate the efficacy of this method for enhancing the production of high-value compounds.

Main Methods:

Developed Multi-targeting Non-specific Genome Engineering (MNGE) using multi-targeting integrase (MTI) systems.
Achieved highly random, multi-copy integration (≥3 copies) requiring only the core TT dinucleotide.
Applied MNGE in Gram-positive (Streptomyces, Saccharopolyspora) and Gram-negative (Burkholderia, Chromobacterium) bacteria.

Main Results:

Successfully integrated fungicide UK-2 BGC (41 kb) and salinomycin BGC (106 kb) into Streptomyces albus, enhancing fermentation levels.
Achieved expression of FR900359 BGC (66 kb) in Burkholderia gladioli using the MTI1 system.
Demonstrated MNGE's broad applicability across diverse bacterial species.

Conclusions:

MNGE is a versatile genome engineering tool for diverse bacteria.
This approach facilitates efficient production of high-value compounds through enhanced gene expression and integration.
MNGE advances next-generation genome engineering for industrial applications.