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A versatile platform strain for high-fidelity multiplex genome editing.

Robert G Egbert1, Harneet S Rishi2,3, Benjamin A Adler4,5

  • 1Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

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Summary

We developed BioDesignER, a novel bacterial strain for enhanced genome engineering. This platform improves recombination efficiency and fidelity, enabling precise genetic modifications for diverse applications.

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Area of Science:

  • Synthetic Biology
  • Microbial Engineering
  • Genomics

Background:

  • Precision genome editing is crucial for understanding genetic traits and engineering organisms.
  • Current recombineering methods often rely on mutator strains with low genomic fidelity and lack regulatory control.
  • High-throughput genetic circuit and pathway engineering requires efficient and accurate genome modification tools.

Purpose of the Study:

  • To develop a new bacterial recombineering platform strain, BioDesignER, with improved efficiency, fidelity, and regulatory control.
  • To overcome the limitations of existing mutator strains used for bacterial genome engineering.
  • To provide a versatile platform for developing and optimizing engineered cellular functions.

Main Methods:

  • Engineered a refactored λ-Red recombination system to reduce toxicity and enhance multi-cycle recombination.
  • Introduced genetic modifications to boost recombination efficiency and fidelity.
  • Integrated four independent inducible transcriptional regulators for precise control of engineered functions.
  • Curated eight context-neutral genomic loci (Safe Sites) for stable gene expression.

Main Results:

  • Achieved single-cycle recombineering efficiencies of up to 25% in the BioDesignER strain.
  • Demonstrated a 7-fold increase in recombineering fidelity compared to the EcNR2 strain.
  • Validated the utility of Safe Sites for consistent gene expression and recombination.

Conclusions:

  • BioDesignER offers a robust and versatile platform for high-throughput bacterial genome engineering.
  • The strain's enhanced fidelity and regulatory control facilitate the development of complex engineered cellular functions.
  • BioDesignER serves as a model for implementing advanced recombination and regulatory systems in other bacterial species.