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Oct 18, 2013

Gene Editing 2.0: Find-and-Replace Feature Works Genome-Wide

  • Scientists have demonstrated techniques that reorder genetic information so thoroughly they have succeeded in creating genomically recoded organisms (GROs). They began by replacing all known UAG stop codons in E. coli with synonymous UAA codons. Not content with this tour de force, they then removed every occurrence of 13 different sense codons across 42 separate E. coli genes and attempted to reshuffle all the remaining codons. When they were done, 24% of the DNA across the 42 targeted genes had been changed. The proteins the genes produced, however, remained identical to those produced by the original genes.

    In assessing the significance of this work, the scientists explained that such extensive genetic engineering could lead to GROs capable of synthesizing proteins that incorporate nonstandard amino acids, offering considerably expanded chemical capabilities. Also, GROs could be engineered to be isolated from nature. Such organisms could better resist viral infections, for example, or be incapable of surviving outside the laboratory.

    The scientists detailed their work in two papers published October 17 in Science. The paper describing the work with the stop codons is entitled "Genomically Recoded Organisms Expand Biological Functions." The paper describing the work with sense codons is entitled "Probing the Limits of Genetic Recoding in Essential Genes."

    Both studies reflect the leadership of George M. Church, Ph.D., the Robert Winthrop Professor of Genetics at Harvard Medical School and founding core faculty member at the Wyss Institute for Biologically Inspired Engineering. Dr. Church’s lab is credited with developing the technologies that made it possible to create the GROs. Among these technologies are the MAGE and CAGE genome editing tools.

    MAGE, or multiplex automated genome engineering, was used to swap out E. coli’s stop codons. Using a piecemeal approach, the scientists created 32 bacterial strains that had only some, not all, of their UAG stop codons substituted with UAA. Every possible substitution, however, was made in at least one of the strains. Then, the bacteria in the different strains were allowed to swap their genes with each other. This swapping was arranged to occur systematically, through the application of CAGE, or conjugative assembly genome engineering.

    Besides using CAGE to arrive at a GRO that had every UAG substituted with a UAA, the scientists removed release factor 1, a protein that recognized the UAG codon and terminates translation. This change was crucial because it permits the reassignment of the UAG codon’s function.

    After assessing their newly created GRO, the scientists said that it exhibited improved properties for incorporation of nonstandard amino acids. Also, it "exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance."

    Moving on from this success, the scientists set the sights even higher. "The first project is saying that we can take one codon, completely remove it from the genome, then successfully reassign its function," said Marc Lajoie, a Harvard Medical School graduate student in the lab of George Church. "For the second project we asked, 'OK, we've changed this one codon, how many others can we change?'"

    Of the 13 codons chosen for the second project, all could be changed. "That leaves open the possibility that we could potentially replace any or all of those 13 codons throughout the entire genome," Lajoie said. The authors of the second paper, however, conceded that while codon usage was malleable, synonymous codons occasionally demonstrated nonequivalence, in unpredictable ways. This result, the authors added, "[underscores] the importance of a strategy that rapidly prototypes and tests many designs in small pieces."

    Together, the two studies suggest not only the power of altering large swaths of genomic material all at once, but the advantages of leveraging the power of natural selection itself. "When an engineering team designs a new cellphone, it’s a huge investment of time and money. They really want that cell phone to work," Dr. Church said. "With E. coli we can make a few billion prototypes with many different genomes, and let the best strain win. That’s the awesome power of evolution."



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