A versatile platform strain for high-fidelity multiplex genome editing
A versatile platform strain for high-fidelity multiplex genome editing
ABSTRACT Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (1) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination efficiency, and (3) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.
Gill Ryan T.、Arkin Adam P.、Adler Benjamin A.、Rishi Harneet S.、Toro Esteban、McCormick Dylan M.、Egbert Robert G.
Department of Chemical and Biomolecular Engineering, University of Colorado BoulderEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory||Department of Bioengineering, University of California - BerkeleyUC Berkeley-UCSF Graduate Program in Bioengineering, University of California - Berkeley||Department of Bioengineering, University of California - BerkeleyBiophysics Graduate Group, University of California - Berkeley||Designated Emphasis Program in Computational and Genomic Biology, University of California - BerkeleyDepartment of Bioengineering, University of California - BerkeleyDepartment of Bioengineering, University of California - BerkeleyEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory
生物工程学遗传学分子生物学
Gill Ryan T.,Arkin Adam P.,Adler Benjamin A.,Rishi Harneet S.,Toro Esteban,McCormick Dylan M.,Egbert Robert G..A versatile platform strain for high-fidelity multiplex genome editing[EB/OL].(2025-03-28)[2025-05-16].https://www.biorxiv.org/content/10.1101/410001.点此复制
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