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Researchers Use Genome Editing Methods to Swap Stop Codons in Living Bacteria

By Andrea Anderson

NEW YORK (GenomeWeb News) – In Science today, an international research group led by investigators at Harvard University and the Massachusetts Institute of Technology reported that they have advanced their genome editing technology, using these tools to develop Escherichia coli strains in which one stop codon has been replaced by another.

"We're able to, at a genome-wide scale, make codon replacements for an entire codon across the whole genome," co-first author Farren Isaacs, who performed the research as a post-doctoral researcher at Harvard University and is currently a researcher at Yale University, told GenomeWeb Daily News. "Basically we use living cells as a template and we make changes within the living cells."

To do this, the team split the E. coli genome into dozens of pieces and then used a method called multiplex automated genome engineering, or MAGE, to introduce codon changes to each region in differents E. coli cultures, trading the guanine nucleotide in the TAG stop codon for an adenine to make the synonymous stop codon TAA. From there, they came up with a hierarchical "conjugative assembly genome engineering" (CAGE) strategy to amalgamate these changes so that increasingly large stretches of the genome were recoded in each intermediate strain.

At the moment, the team has four E. coli strains that they plan to use to generate a single strain in which every TAG has been converted to TAA.

"These four strains, which contain up to 80 modifications per genome, can be combined to complete the assembly of a fully recoded strain containing all 314 TAG-to-TAA codon conversions," the study authors wrote.

The long-term objectives of such experiments are to develop the technology to make large-scale changes to genomes and introduce new functions into organisms, Isaacs explained, and, eventually, to create organisms with new genetic codes, including those capable of producing proteins from amino acids not currently found in nature.

"That could lead to entirely new classes of drugs, industrial enzymes, biopolymers that could be used to make new types of materials, and so on," he said, noting that similar strategies could also be used to genetically isolate organisms, thwarting potential viral pathogens, and to contain genetically engineered organisms within restricted environments.

Members of the team previously used MAGE to reprogram a biosynthesis pathway in E. coli cells leading to enhanced production of the compound lycopene — work that they reported in Nature in 2009.

Now, researchers have shown that they can build on this method, putting together pieces of the genome containing MAGE-induced changes to produce bacterial strains with specific codon changes across larger and larger swaths of the genome.

The 20 amino acids and "stop" signal are encoded by 64 triplet nucleotide sequences, meaning there are more than three times as many codons as there are functions for which they code.

The researchers were able to exploit this redundancy in the genetic code, Isaacs explained, trading the stop codon TAG, which usually appears 314 times in the E. coli genome, for another stop codon, TAA, that's recognized by a different release factor during translation.

The team first introduced targeted alterations into 32 E. coli cultures by MAGE using oligonucleotides containing the desired changes, Isaacs said, gauging the functional consequences, if any, along the way.

"We decided to pursue a strategy whereby we would divide up the strains and quickly introduce all of these changes to small pools to, one, verify that they're viable and, two, be able to detect and quantify phenotypes," he explained. "It was important to be able to obtain enough resolution on the changes that we're making to see if any of them led to any sort of strange phenotype."

They then merged the MAGE-produced alterations into their final four intermediate strains using CAGE. Coupled with selection, this hierarchical genome engineering method let the researchers transplant well-defined stretches of DNA from one genome to another without introducing unintended changes to the recipient genome.

The researchers are now continuing to use CAGE to take the E. coli strains with the most extensive recoding to the next stage — assembling a strain in which every TAG in the genome has been converted to TAA.

In the future, they also plan to try trading out other codons, including those coding for amino acids. The team will likely attempt to use their codon replacement strategy in other bacterial species, Isaacs noted, and, perhaps, in eukaryotic cells as well.

"Our methods treat the chromosome as both an editable and an evolvable template," the researchers wrote, "permitting the exploration of vast genetic landscapes."