Article | Published:

Defining synonymous codon compression schemes by genome recoding

Nature volume 539, pages 5964 (03 November 2016) | Download Citation

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.

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Acknowledgements

Work was supported by the Medical Research Council, UK (MC_U105181009 and MC_UP_A024_1008, J.W.C.), the Danish Council for Independent Research (DFF – 4090-00289, to J.F.), a Boehringer Ingelheim Fonds PhD fellowship (to S.F.B.), The Gates-Cambridge Scholarship (to S.H.K), and an ERC Advanced Grant (SGCR to J.W.C.). We thank Neil Grant MRC-LMB Visual Aids for photography.

Author information

Author notes

    • Julius Fredens
    •  & Simon F. Brunner

    These authors contributed equally to this work.

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Kaihang Wang
    • , Julius Fredens
    • , Simon F. Brunner
    • , Samuel H. Kim
    • , Tiongsun Chia
    •  & Jason W. Chin
  2. Department of Chemistry, Cambridge University, Cambridge CB2 1EW, UK

    • Samuel H. Kim
    •  & Jason W. Chin

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Contributions

J.W.C. defined the direction of research. K.W. designed and constructed the REXER and GENESIS systems. J.F. implemented DNA assembly methods in S. cerevisiae. K.W. and S.F.B. identified target codons, developed tE and designed recoding schemes. K.W., J.F., S.F.B., S.H.K., and T.C. performed experiments. All authors analysed the data. K.W. and J.W.C. wrote the paper with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Kaihang Wang or Jason W. Chin.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains original DNA gel images.

Text files

  1. 1.

    Supplementary Data 1

    Genomic locus for selection marker -1/+1.

  2. 2.

    Supplementary Data 2

    Plasmid containing lambda red, Cas9, and tracrRNA.

  3. 3.

    Supplementary Data 3

    BAC containing lux operon and -2/+2 for integration.

  4. 4.

    Supplementary Data 4

    Plasmid containing spacers for REXER 2.

  5. 5.

    Supplementary Data 5

    Plasmid containing spacers for REXER 4.

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DOI

https://doi.org/10.1038/nature20124

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