title: Defining synonymous codon compression schemes by genome recoding.
abstract: Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.
authors: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors: Kaihang
authors: K
authors: Wang
authors: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors: Julius
authors: J
authors: Fredens
authors: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors: Simon F
authors: SF
authors: Brunner
authors: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.|Department of Chemistry, Cambridge University, Cambridge, England, UK.
authors: Samuel H
authors: SH
authors: Kim
authors: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors: Tiongsun
authors: T
authors: Chia
authors: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.|Department of Chemistry, Cambridge University, Cambridge, England, UK.
authors: Jason W
authors: JW
authors: Chin
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics; D003062:Codon / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D020043:Genes, Essential / Q000235:genetics; D005815:Genetic Code / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial / Q000235:genetics*; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012641:Selection, Genetic; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D003062:Codon; D004247:DNA