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[Optimization of base editing in Corynebacterium glutamicum].

Junwei Li1,2, Ye Liu2,3, Yu Wang2,3

  • 1School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457, China.

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology
|February 20, 2020
PubMed
Summary

We optimized CRISPR/Cas9 base editing in Corynebacterium glutamicum using a GFP reporter system. Optimized conditions significantly improved base editing efficiency by up to 2.5-fold in endogenous loci.

Keywords:
CRISPR/Cas systemCorynebacterium glutamicumbase editingediting condition

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Microbial Genetics

Background:

  • CRISPR/Cas9 base editing is a powerful genome editing tool without double-stranded DNA breaks.
  • Multiplex automated base editing with chemical control (MACBETH) was previously developed for Corynebacterium glutamicum.
  • Improving base editing efficiency in industrial microbial strains is crucial for genetic engineering.

Purpose of the Study:

  • To optimize base editing conditions in Corynebacterium glutamicum.
  • To establish a rapid and accurate method for assessing base editing efficiency.
  • To enhance the efficiency and versatility of base editing in industrial applications.

Main Methods:

  • Construction of a green fluorescent protein (GFP) reporter system for detecting base editing events.
  • Utilizing flow cytometry for high-throughput quantification of base editing efficiency.
  • Systematic optimization of medium, induction OD₆₀₀, induction time, and IPTG concentration.
  • Validation of optimized conditions on endogenous genomic loci.

Main Results:

  • A GFP reporter system coupled with flow cytometry allowed for rapid efficiency calculation.
  • Initial base editing efficiency was 13.11±0.21%.
  • Optimized conditions (CGXII medium, 0.05 OD₆₀₀, 20h induction, 0.01 mmol/L IPTG) increased efficiency to 30.35±0.75% (1.3-fold).
  • Optimized conditions enhanced editing efficiency 1.7-2.5 fold at endogenous loci.

Conclusions:

  • The optimized base editing protocol significantly enhances editing efficiency in Corynebacterium glutamicum.
  • The developed GFP reporter system provides a robust platform for base editing optimization.
  • This improved method holds promise for broader applications of base editing in industrial microbiology.