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July 15, 2011

New technology from MIT and Harvard can edit DNA at the genome scale

MIT and Harvard researchers have developed technologies that could be used to rewrite the genetic code of a living cell, allowing them to make large-scale edits to the cell’s genome. Such technology could enable scientists to design cells that build proteins not found in nature, or engineer bacteria that are resistant to any type of viral infection.

MIT Technology Review - George Church says he hopes to achieve three goals with the approach. First, he wants to build bacteria that can produce new drugs and other chemicals. Second, he wants to genetically engineer bacteria that cannot live outside the lab because they need unnatural amino acids to survive—a feat that could prevent the environmental damage that might result from such bacteria being let loose in the world. And third, he wants to make bacteria that are immune to viruses, since viruses can cause problems in industrial production. "The way to achieve all these things is to change the [meaning of the] genetic code of your favorite organism," says Church.

In the journal Science, Church's group described how it deleted all 314 instances of a particular codon in the genome of living E. coli and replaced them with another codon.

The researchers combined a technique they previously unveiled in 2009, called multiplex automated genome engineering (MAGE), with a new technology dubbed conjugative assembly genome engineering (CAGE).

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



We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) 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. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.

90 pages of supplemental material

Church's method introduces changes in living cells. He believes the advantage of this approach is that it's possible to correct mistakes as they happen on the way toward making larger changes. Church hopes his latest work will convince other researchers of the value of "genome-scale" engineering. Both his method and that developed at the Venter Institute involve using DNA synthesizer machines to make large amounts of DNA for the engineered cells to take up. DNA synthesis is still expensive. And the time involved in both techniques, though it's getting shorter, is another expense. "We need to bring costs down, and think about ease of use," he says.

Two companies—Allozyme, which Tirrell is associated with, and Ambrix—are both making protein drugs that incorporate unnatural amino acids. In both cases, they have engineered bacteria that can make proteins that include just one unnatural amino acid. Making organisms that can use more of these unnatural chemicals to produce new kinds of molecules would open up new frontiers for protein drugs, he says. Proteins with unnatural components might also be able to cross barriers in the body that are not easily breached today, such as the blood-brain barrier. Church's group is beginning a collaboration with Ambrix.

Because the alterations were done in living cells, the researchers have been able to monitor any potential harmful effects as they appear and current results suggested that the final four strains were healthy, and can survive and reproduce.

The researchers are confident that they will create a single strain in which all TAG codons are eliminated, after which they plan to delete the cell machinery that reads the TAG condon to free it up for a completely new purpose, such as encoding a novel amino acid.

In addition to adding functionality to a cell by encoding for useful new amino acids, George Church, professor of genetics at Harvard Medical School, says the technology could also be used to introduce safeguards that prevent cross-contamination between modified organisms and the wild. Additionally, it could be used to establish multi-viral resistance by rewriting code hijacked by viruses. This would be of particular interest to industries that cultivate bacteria, such as the pharmaceuticals and energy industries, where such viruses affect up to 20 percent of cultures resulting in losses in the billions of dollars.

Altering the genetic code of industrial bacteria could also create a “genetic firewall” that would prevent engineered bacteria from spreading their genes to natural bacteria in the environment, or from allowing such bacteria to survive in the wild, Carr says.

Prior work

Accelerated evolution machine being adapted to human stem cells and creates the new field of Recombineering

* Church is adapting MAGE for genetically modifying human stem cell lines. The work, funded by the US National Human Genome Research Institute, aims to create human cell lines with subtly different genomes in order to test ideas about which mutations cause disease and how.

* if MAGE really can be used to edit the genome of human cells, it would provide a way to fix the mutations that cause inherited disease. It could be the technology that opens the door to the genetic engineering of humans.

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