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Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS

Robertson, Wesley E. and Funke, Louise F. H. and de la Torre, Daniel and Fredens, Julius and Wang, Kaihang and Chin, Jason W. (2021) Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS. Nature Protocols, 16 (5). pp. 2345-2380. ISSN 1754-2189. doi:10.1038/s41596-020-00464-3.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription ReadCube access ItemCode used for next-generation sequencing analysis
Robertson, Wesley E.0000-0001-9812-8288
de la Torre, Daniel0000-0002-9545-9388
Fredens, Julius0000-0002-4262-9304
Wang, Kaihang0000-0001-7657-8755
Chin, Jason W.0000-0003-1219-4757
Additional Information:© 2021 Nature Publishing Group. Received 02 May 2019. Accepted 16 November 2020. Published 26 April 2021. 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. These authors contributed equally: Wesley E. Robertson, Louise F. H. Funke, Daniel de la Torre, Julius Fredens. Author Contributions. All authors contributed to developing this protocol and writing this paper. J.W.C. supervised the project. 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 The authors declare no competing interests. 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.
Funding AgencyGrant Number
Medical Research Council (UK)MC_U105181009
Medical Research Council (UK)MC_UP_A024_1008
Medical Research FoundationMRF-109-0003-RG-CHIN/C0741
European Research Council (ERC)UNSPECIFIED
Lundbeck FoundationR232-2016-3474
Subject Keywords:Genetic engineering; Genomic engineering; Synthetic biology
Issue or Number:5
Record Number:CaltechAUTHORS:20201030-111758974
Persistent URL:
Official Citation: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).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106358
Deposited By: George Porter
Deposited On:26 Apr 2021 21:17
Last Modified:16 Nov 2021 18:53

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