Combining MAGE with CAGE allowed the genome to manipulated as an editable and evolvable template.

Scientists have combined a recently developed technique known as multiplex automated genome engineering (MAGE) with a newly developed platform, hierarchical conjugative assembly genome engineering (CAGE), to site-specifically replace all 314 TAG stop codons in E. coli with a synonymous TAA codon. The overall process combines synthetic DNA and recombination methods to introduce genome-wide changes dynamically in living cells, effectively engineering the genome through viable intermediates.

Describing their E. coli  manipulation in Science, the collaborators at Harvard Medical School and MIT, say the approach also allowed them to measure individual recombination frequencies, confirm cell viability at each modification, track additional mutations, and identify associated phenotypes.

“We present genome-engineering technologies that are capable of fundamentally re-engineering genomes from the nucleotide to the megabase scale,” the authors state. Harvard’s George M. Church, Ph.D., and Farren  J. Isaacs, Ph.D., together with MIT’s Peter A. Carr, Ph.D., and colleagues, describe their achievements in a paper titled “Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement.”

First described by the Harvard and MIT researchers in 2009, MAGE is a continuous, cyclical technique that enables the rapid generation of sequence diversity at numerous targeted chromosomal locations across a large population of cells through the repeated introduction of synthetic DNA. In the current set of experiments, the technique was used as the first stage in a process that ultimately aimed to generate a strain of E. coli in which every TAG stop codon was replaced by a TAA codon.

The researchers first split the E. coli genome into 32 regions, each carrying 10 TAG codons, and used MAGE to replace 10 TAG codons with TAA codons in each of bacterial strains. This resulted in a series of engineered bacteria that together included every TAG-TAA replacement.

The team then used CAGE to effect hierarchical assembly of the modified chromosomal segments into a single strain. CAGE exploits bacterial conjugation, the method by which bacteria transfer genes to each other. However, in contrast with natural mechanisms of conjugal DNA transfer, CAGE allows researchers to precisely control the genomic position at which conjugal transfer is initiated.

They divided their 32 MAGE-modified E. coli strains into 16 pairs for conjugation. Within each pair, a donor strain transferred its recoded genomic region to the recipient strain. This recipient effectively then carried the donated region (with 10 TAG-TAA replacements) as well as its own MAGE-recoded region that carried a different set of 10 TAG-TAA replacements.

This first stage of CAGE resulted in 16 strains each carrying 20 modifications. The 16 strains were then separated into eight pairs, and the controlled conjugation repeated, resulting in eight stains each carrying 40 modifications.

The aim was to continue the process of pairing plus conjugation until a single strain carrying each of the 314 TAG-TAA replacements was generated. However, the Stanford and MIT team’s publication describes the results up until the point at which there were four groups of healthy bacteria, each containing 80 TAG-TAA changes.

“This study, which integrates in vivo genome engineering from the nucleotide to the megabase scale, demonstrates the successful replacement of all genomic occurrences of the TAG stop codon in the E. coli genome,” the authors summarize. “We found that cells can incorporate all individual TAG-to-TAA codon changes, and that these changes can be assembled into genomes with up to 80 modifications with mild phenotypic consequences.

“In contrast to in vitro genome synthesis and transplantation methods that introduce discrete and abrupt changes in a single genome, our genome engineering technologies treat the chromosome as an editable and evolvable template and generate targeted and combinatorial modifications across many genomes in vivo.”

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