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MACBETH: Multiplex automated Corynebacterium glutamicum base editing method.

Yu Wang1, Ye Liu1, Jiao Liu1

  • 1Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.

Metabolic Engineering
|March 28, 2018
PubMed
Summary
This summary is machine-generated.

We developed a multiplex automated Corynebacterium glutamicum base editing method (MACBETH) for efficient bacterial genome editing without foreign DNA templates. This automated CRISPR-based system significantly enhances metabolic engineering and strain development for industrial applications.

Keywords:
CRISPR/Cas9Corynebacterium glutamicumCytidine deaminaseGenome editingMultiplex automated base editing

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Biotechnology

Background:

  • CRISPR/Cas9 and Cpf1-induced double-strand breaks reduce bacterial survival, limiting multiplex genome editing.
  • Reliance on foreign DNA templates for each target locus is labor-intensive and raises regulatory concerns for industrial use.

Purpose of the Study:

  • To develop a multiplex automated bacterial base editing method for efficient genome engineering.
  • To overcome limitations of traditional CRISPR methods in bacteria, particularly for industrial applications.

Main Methods:

  • Developed the multiplex automated Corynebacterium glutamicum base editing method (MACBETH) using CRISPR/Cas9 and activation-induced cytidine deaminase (AID).
  • Implemented MACBETH without foreign DNA templates for single-, double-, and triple-locus editing.
  • Automated the MACBETH system with an integrated robotic platform for high-throughput strain engineering.

Main Results:

  • Achieved high editing efficiencies: up to 100% for single-locus, 87.2% for double-locus, and 23.3% for triple-locus editing.
  • Generated a gene inactivation library to improve glutamate production, with a pyk&ldhA double inactivation strain showing a 3-fold increase.
  • Successfully constructed a genome-scale library of 94 transcription factors with 100% success rate using the automated platform.

Conclusions:

  • MACBETH offers a powerful, template-free solution for multiplex and automated bacterial genome editing.
  • The automated system enables the generation of thousands of engineered strains monthly, accelerating metabolic engineering.
  • MACBETH is poised to be a transformative tool for both research and industrial applications in bacterial engineering.