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Defining synonymous codon compression schemes by genome recoding

Wang, Kaihang and Fredens, Julius and Brunner, Simon F. and Kim, Samuel H. and Chia, Tiongsun and Chin, Jason W. (2016) Defining synonymous codon compression schemes by genome recoding. Nature, 539 (7627). pp. 59-64. ISSN 0028-0836. PMCID PMC5321499. doi:10.1038/nature20124. https://resolver.caltech.edu/CaltechAUTHORS:20180626-165021917

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[img] Image (JPEG) (Extended Data Figure 1 : Simultaneous double selection and recombination enhances integration at a target locus) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 2 : REXER enables site-specific integration of large DNA fragments into the genome) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 3 : Replacement of 100 kb of genomic DNA via REXER) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 4 : Iterative REXER) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 5 : Synonymous codon compression strategies) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 6 : Recoding landscapes for compression of serine codons by REXER) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 8 : Identifying and fixing a deleterious sequence in defined and systematic synonymous recoding) - Supplemental Material
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[img] Image (JPEG) (Extended Data Table 1: Defining recoding rules by codon adaptation index (cAi), tRNA adaptation index (tAi), and translation efficiency (tE)) - Supplemental Material
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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/nature20124DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321499/PubMed CentralArticle
https://rdcu.be/1XSMPublisherFree ReadCube access
ORCID:
AuthorORCID
Wang, Kaihang0000-0001-7657-8755
Fredens, Julius0000-0002-4262-9304
Chin, Jason W.0000-0003-1219-4757
Additional Information:© 2016 Macmillan Publishers Limited. Received 11 February; accepted 26 September 2016. Published online 24 October 2016. 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. Julius Fredens & Simon F. Brunner: These authors contributed equally to this work. Author 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. The authors declare no competing financial interests. Data availability: The sequences used in this study are available in Supplementary Data 1–5. Supplementary Data 1: Genomic locus for selection marker −1/+1; Supplementary Data 2: Plasmid containing lambda red, Cas9, and tracrRNA; Supplementary Data 3: BAC containing lux operon and −2/+2 for integration; Supplementary Data 4: Plasmid containing spacers for REXER 2; Supplementary Data 5: Plasmid containing spacers for REXER 4. All other datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.
Funders:
Funding AgencyGrant Number
Medical Research Council (UK)MC_U105181009
Medical Research Council (UK)MC_UP_A024_1008
Danish Council for Independent ResearchDFF – 4090-00289
Boehringer Ingelheim FondsUNSPECIFIED
Gates-Cambridge ScholarshipUNSPECIFIED
European Research Council (ERC)UNSPECIFIED
Issue or Number:7627
PubMed Central ID:PMC5321499
DOI:10.1038/nature20124
Record Number:CaltechAUTHORS:20180626-165021917
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180626-165021917
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:87368
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:27 Jun 2018 14:34
Last Modified:15 Nov 2021 20:47

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