title: Precise manipulation of chromosomes in vivo enables genome-wide codon replacement.
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.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. farren.isaacs@yale.edu
authors.forename: Farren J
authors.initials: FJ
authors.lastname: Isaacs
authors.forename: Peter A
authors.initials: PA
authors.lastname: Carr
authors.forename: Harris H
authors.initials: HH
authors.lastname: Wang
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Lajoie
authors.forename: Bram
authors.initials: B
authors.lastname: Sterling
authors.forename: Laurens
authors.initials: L
authors.lastname: Kraal
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Tolonen
authors.forename: Tara A
authors.initials: TA
authors.lastname: Gianoulis
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Goodman
authors.forename: Nikos B
authors.initials: NB
authors.lastname: Reppas
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Emig
authors.forename: Duhee
authors.initials: D
authors.lastname: Bang
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Hwang
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jacobson
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D018388:Codon, Terminator; D003227:Conjugation, Genetic; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D042822:Genomic Instability; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011995:Recombination, Genetic; D013698:Templates, Genetic
chemical_list: D018388:Codon, Terminator