Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS
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1.
MRC Laboratory of Molecular Biology
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2.
California Institute of Technology
Abstract
We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7–10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR–Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.
Copyright and License
© 2021, The Author(s), under exclusive licence to Springer Nature Limited
Acknowledgement
This work was supported by the Medical Research Council (MRC), UK (MC_U105181009 and MC_UP_A024_1008), the Medical Research Foundation (MRF-109-0003-RG-CHIN/C0741) and an ERC Advanced Grant SGCR, all to J.W.C., and by the Lundbeck Foundation (R232-2016-3474) to J.F. K.W. was supported by the NIH DP2 Award GM140937. J.W.C. thanks H. Pelham for supporting this project. We are grateful to the LMB Media Kitchen for help with preparing materials.
Contributions
All authors contributed to developing this protocol and writing this paper. J.W.C. supervised the project. These authors contributed equally: Wesley E. Robertson, Louise F. H. Funke, Daniel de la Torre, Julius Fredens.
Software References
Code used for next-generation sequencing analysis is freely available at https://github.com/TiongSun/iSeq.
Errata
21 November 2024. A Correction to this paper has been published: https://doi.org/10.1038/s41596-024-01114-8.
In the version of the article initially published, the sentence “K.W. was supported by the NIH DP2 Award GM140937” was missing from the Acknowledgements and has now been added to the HTML and PDF versions of the article.
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Additional details
- Medical Research Council
- MC_U105181009
- Medical Research Council
- MC_UP_A024_1008
- Medical Research Foundation
- MRF-109-0003-RG-CHIN/C0741
- Lundbeck Foundation
- R232-2016-3474
- National Institutes of Health
- DP2 Award GM140937
- Accepted
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2020-11-16Accepted
- Available
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2021-04-26Published online
- Caltech groups
- Division of Biology and Biological Engineering
- Publication Status
- Published