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Related Experiment Video

Updated: Nov 1, 2025

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

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Published on: February 28, 2025

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.

Yiwen Zhang^1,2, Jiawei Yang^1,2, Siqi Yang^1,2

¹Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China; Shanghai Institutes for Biological Sciences, Huzhou, China.

The CRISPR Journal

|June 21, 2021

Summary

We developed MUCICAT, a CRISPR-based tool for multiplex gene editing in Escherichia coli, enabling rapid creation of diverse genetic variants. This system efficiently engineered N-acetylglucosamine production, achieving high yields in just 8 days.

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

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Area of Science:

Synthetic biology
Microbial engineering
Genomics

Background:

Directed evolution and genome editing are powerful but limited in scale.
Current methods often require high-throughput screening or single-gene manipulation.
Simultaneous, multiplex genetic modification is needed for complex engineering.

Purpose of the Study:

To develop a novel CRISPR-associated transposase system for multiplex chromosomal integration.
To engineer the N-acetylglucosamine (GlcNAc) biosynthesis pathway in Escherichia coli for enhanced production.
To create combinatorial genomic diversity for strain optimization.

Main Methods:

Implementation of multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT).
Optimization of the MUCICAT system by modifying promoter and transposase expression.
Application of MUCICAT for multiplex gene insertion of the GlcNAc cassette into E. coli.

Main Results:

MUCICAT successfully targeted up to 11 sites simultaneously for gene interruption and insertion.
Engineered E. coli produced 11.59 g/L of GlcNAc, a sixfold increase compared to a control strain.
A variant with five copies of the GlcNAc cassette demonstrated superior production.

Conclusions:

MUCICAT is a powerful tool for generating combinatorial genomic diversity and cell programming.
The system enables efficient engineering of metabolic pathways for industrial applications.
MUCICAT has broad potential applications in synthetic biology and beyond.