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

Author

Massachusetts Institute of Technology. Center for Bits and Atoms, Massachusetts Institute of Technology. Media Laboratory, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Materials Science and Engineering, Isaacs, Farren J, Carr, Peter A, Wang, Harris H, Lajoie, Marc J, Sterling, Bram, Kraal, Laurens, Tolonen, Andrew C, Gianoulis, Tara A, Goodman, Daniel B, Reppas, Nikos B, Emig, Christopher J, Bang, Duhee, Hwang, Samuel J, Jewett, Michael C, Jacobson, Joseph M, Church, George M, Massachusetts Institute of Technology. Center for Bits and Atoms, Massachusetts Institute of Technology. Media Laboratory, Harvard University--MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology. Department of Materials Science and Engineering, Isaacs, Farren J, Carr, Peter A, Wang, Harris H, Lajoie, Marc J, Sterling, Bram, Kraal, Laurens, Tolonen, Andrew C, Gianoulis, Tara A, Goodman, Daniel B, Reppas, Nikos B, Emig, Christopher J, Bang, Duhee, Hwang, Samuel J, Jewett, Michael C, Jacobson, Joseph M, and Church, George M

Abstract

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.

Published
2021

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