Redesigned genomes require approaches that reconcile the desired biological behavior with challenges inherent to biological complexity. Engineering biological systems can be unpredictable, as a single misplaced or misdesigned allele can be lethal. To address these challenges, we have developed approaches that integrate synthetic DNA and recombination methods to introduce genome-wide changes dynamically in living cells, thereby engineering the genome through viable intermediates. In recent work, we developed multiplex automated genome engineering (MAGE), which rapidly generates genetic diversity for strain and pathway engineering. To augment MAGE’s ability to introduce nucleotide scale mutations across the genome, a complementary method was required to assemble modified chromosomes in vivo.

Here, we report the development of a hierarchical conjugative assembly genome engineering (CAGE) method and its integration with MAGE toward reengineering the canonical genetic code of E. coli—an organism with broad utility in basic and applied research.

—Isaacs et al.

In the paper, the researchers describe how they replaced instances of a codon—a DNA “word” of three nucleotide letters—in 32 strains of E. coli, and then coaxed those partially edited strains along an evolutionary path toward a single cell line in which all 314 instances of the codon had been replaced.

That many edits surpasses current methods by two orders of magnitude, said Harris Wang, a research fellow in Church’s lab at the Wyss Institute for Biologically Inspired Engineering who shares lead-author credit on the paper with Farren Isaacs, an assistant professor of molecular, cellular, and developmental biology at Yale University and a former Harvard research fellow, and Peter Carr, a research scientist at the MIT Media Lab.

In the genetic code, most codons specify an amino acid, a protein building block. But a few codons tell the cell when to stop adding amino acids to a protein chain, and it was one of these “stop” codons that the Harvard researchers targeted. With just 314 occurrences, the TAG stop codon is the rarest word in the E. coli genome, making it a prime target for replacement.

Using MAGE, the team replaced instances of the TAG codon with another stop codon, TAA, in living E. coli cells. (Unveiled by the team in 2009, the MAGE process has been called an evolution machine for its ability to accelerate targeted genetic change in living cells.)

While MAGE, a small-scale engineering process, yielded cells in which TAA codons replaced some but not all TAG codons, the team constructed 32 strains that, taken together, included every possible TAA replacement. Then, using bacteria’s innate ability to trade genes through a process called conjugation, the researchers induced the cells to transfer genes containing TAA codons at increasingly larger scales.

Strategy for reassigning all 314 TAG codons to TAA in E. coli. First, the genome was split into 32 regions each containing 10 TAG stop codons. In parallel, MAGE was used to execute all 10 TAG::TAA codon modifications in a single strain for each genomic region. These partially recoded strains were paired such that a targeted genomic region of one strain (donor) was strategically transferred into a second strain (recipient), permitting the hierarchical consolidation of modified genomic regions using CAGE. This five-stage process transfers genomic fragments ranging in size from ~154 kb to ~2.3 Mb in a controlled manner until a single recoded strain is constructed that lacks the TAG stop codon throughout. Once all TAG codons have been converted to TAA, the prfA gene will be deleted to inactivate TAG translational termination. Isaacs et al. Click to enlarge.

The new method—conjugative assembly genome engineering (CAGE)—resembles a playoff bracket—a hierarchy that winnows 16 pairs to eight to four to two to one—with each round’s winner possessing more TAA codons and fewer TAG, explains Isaacs.

We’re testing decades-old theories on the conservation of the genetic code. And we’re showing on a genome wide scale that we’re able to make these changes.

—Farren Isaacs

Church said that the ability to make such functional and radical changes serves three goals:

• Adding functionality to a cell by encoding for useful new amino acids;
• Introducing safeguards that prevent cross-contamination between modified organisms and the wild; and
• Establishing multiviral resistance by rewriting code hijacked by viruses. In industries that cultivate bacteria, including pharmaceuticals and energy, such viruses affect up to 20% of cultures. A notable example afflicted the biotech company Genzyme, where estimates of losses due to viral contamination range from a few hundred million dollars to more than $1 billion.

The team published their results with 28 of 31 conjugations having been completed. Results suggested that the final four strains were healthy, even as the team assembled four groups of 80 engineered alterations into stretches of the chromosome surpassing 1 million DNA base pairs.

The researchers are confident that they will create a single strain in which TAG codons are completely eliminated. The next step, they say, is to delete the cell’s machinery that reads the TAG gene—freeing up the codon for a completely new purpose, such as encoding a novel amino acid.

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. 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. The scarless introduction of codon changes via MAGE enabled the first genome-wide allelic replacement frequency map using single-stranded DNA oligos in E. coli. In addition, our engineered conjugation experiments produced a complementary recombination frequency map of intergenic dsDNA integration sites across the genome.

Together, these experiments revealed both highly accessible and recalcitrant sites for both small- and large-scale chromosomal modifications. These data could serve as valuable resources for future genome engineering efforts. Moreover, synthetic approaches such as the one pursued here may help to refine the existing genome annotation by revealing unannotated functional genetic loci, such as short peptides or minigenes.

Introducing genome-wide changes dynamically in a living cell permits engineering in the cell’s native biological context. 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 (~10⁹) genomes in vivo.

MAGE is optimal for introducing small modifications in sequence design space, whereas CAGE is designed for taking bigger leaps via large-scale assembly of many modified genomes. Together, these genome editing methods are advantageous when the designed genomes share >90% sequence similarity to existing templates or when many targeted mutations dispersed across the chromosome are desired (e.g., genome recoding).

—Isaacs et al.

This research was funded by US Department of Energy and the National Science Foundation.

Resources

• Farren J. Isaacs, Peter A. Carr, Harris H. Wang, Marc J. Lajoie, Bram Sterling, Laurens Kraal, Andrew C. Tolonen, Tara A. Gianoulis, Daniel B. Goodman, Nikos B. Reppas, Christopher J. Emig, Duhee Bang, Samuel J. Hwang, Michael C. Jewett, Joseph M. Jacobson, and George M. Church (2011) Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement. Science 333 (6040), 348-353. doi: 10.1126/science.1205822