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Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS


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.

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Fig. 1: Schematic of REXER and GENESIS.
Fig. 2: BAC assembly and delivery.
Fig. 3: Components for REXER.
Fig. 4: REXER workflow.
Fig. 5: Identifying deleterious sequences in synthetic DNA.
Fig. 6: Testing potential fixes for deleterious synthetic sequences.

Data availability

No new data were generated or analyzed while preparing this protocol. Representative data are available in refs. 3,14.

Code availability

Code used for next-generation sequencing analysis is freely available at


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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. J.W.C. thanks H. Pelham for supporting this project. We are grateful to the LMB Media Kitchen for help with preparing materials.

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Authors and Affiliations



All authors contributed to developing this protocol and writing this paper. J.W.C. supervised the project.

Corresponding authors

Correspondence to Kaihang Wang or Jason W. Chin.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Byung-Kwan Cho, Bogumil Karas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Wang, K. et al. Nature 539, 59–64 (2016):

Fredens, J. et al. Nature 569, 514–518 (2019):

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Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Primers used in this study.

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Robertson, W.E., Funke, L.F.H., de la Torre, D. et al. Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS. Nat Protoc 16, 2345–2380 (2021).

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