Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA

  title={Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA},
  author={Ryan R. Gallagher and Zhe Li and Aaron O Lewis and Farren J. Isaacs},
  journal={Nature Protocols},
Multiplex automated genome engineering (MAGE) is a powerful technology for in vivo genome editing that uses synthetic single-stranded DNA (ssDNA) to introduce targeted modifications directly into the Escherichia coli chromosome. MAGE is a cyclical process that involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. By iteratively introducing libraries of… 

Strategies to Identify and Edit Improvements in Synthetic Genome Segments Episomally

Improvements to a multiplex automated genome engineering (MAGE) protocol are described to improve recombineering frequency and multiplexability and could be adapted to mutation identification and repair for other large-scale genome engineering projects, or for incorporation of small genetic engineering sites for quick protocol adjustments.

Recombineering and MAGE.

This Primer describes recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools, and looks forward to the future of genetic engineering.

Systematic genome engineering approaches to investigate mutational effects and evolutionary processes

DIvERGE offers a versatile solution for high-precision directed evolution at multiple loci in their native genomic context and is especially well-suited to study bacterial evolution leading to antibiotic resistance.

Retroelement-Based Genome Editing and Evolution.

This coupled mutagenic T7 RNA polymerase-retron system enabled the evolution of diverse variants of environmentally selected antibiotic resistance genes, producing mutation rates in the targeted region 190-fold higher than background cellular mutation rates, potentially enabling the dynamic, continuous self-evolution of selected phenotypes.

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli

It is argued that the pre-selection rule of genome-library-optimized-sequences (GLOS) allows for stable and efficient fine tuning of chromosomal functions with minimal effort and can be used in complex genome editing operations such as concomitant deletions.

Genome-Wide Abolishment of Mobile Genetic Elements Using Genome Shuffling and CRISPR/Cas-Assisted MAGE Allows the Efficient Stabilization of a Bacterial Chassis.

The combined capacity of phage-mediated generalized transduction and CRISPR/Cas-selected MAGE offers a way for rapid, large scale editing of bacterial genomes.

Targeted editing and evolution of engineered ribosomes in vivo by filtered editing.

Genome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered

Toward Genome-Based Metabolic Engineering in Bacteria.

Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein

The results suggest that an ancient–modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes.



Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

Removing a set of five exonucleases substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE) and investigates and clarify the effects of oligonucleotide phosphorothioation on recombination frequency.

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides

It is demonstrated in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs as short as 30 bases long, which provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes.

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

  • N. CostantinoD. Court
  • Biology
    Proceedings of the National Academy of Sciences of the United States of America
  • 2003
The results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.

Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement

H hierarchical conjugative assembly genome engineering (CAGE) was developed to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects.

Programming cells by multiplex genome engineering and accelerated evolution

The multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties by facilitating rapid and continuous generation of a diverse set of genetic changes.

Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion

A novel strategy using oligos containing chemically modified bases in place of the standard T, C, A or G to avoid mismatch detection and repair is presented, which increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level.

Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering

Improvements in MAGE will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening, and both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE.

Conditional DNA repair mutants enable highly precise genome engineering

A novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli is presented, which reduces the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement.

Genome engineering with targetable nucleases.

  • D. Carroll
  • Biology
    Annual review of biochemistry
  • 2014
Three classes of targetable cleavage reagents are described: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nuclease (RGNs), which have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans.