ArticleNature protocols2021
Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS.
Article in Nature protocols, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- Bridging continuous and discrete evolution through a controllable, hypermutagenic phage-bacteria system.Nature microbiology · 2026Article
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- Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences.Nature communications · 2026Article
- Synthetic Biology of Plants and Microbes for Agriculture, Environment, and Future Applications.Chemical reviews · 2026Review
- Synthetic rewriting technologies in mammalian cells.Nature communications · 2026Review
- Recent technology advancements in large-scale DNA assembly.Quantitative biology (Beijing, China) · 2025Article
- Modulating bacterial function utilizing A knowledge base of transcriptional regulatory modules.Nucleic acids research · 2024Article
- Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids.Chemical reviews · 2024Review
- Advancing the scale of synthetic biology via cross-species transfer of cellular functions enabled by iModulon engraftment.Nature communications · 2024Article
- Recent advances in genome-scale engineering inEngineering microbiology · 2024Review
- Investigation of Genome Biology by Synthetic Genome Engineering.Bioengineering (Basel, Switzerland) · 2023Review
- Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles.Nature chemistry · 2023Article
- Current trends in biopharmaceuticals production in Escherichia coli.Biotechnology letters · 2022Review
- Overlapping genes in natural and engineered genomes.Nature reviews. Genetics · 2022Review
- Strategies to Enhance Periplasmic Recombinant Protein Production Yields inFrontiers in bioengineering and biotechnology · 2021Review
Corrections and comments
- Erratum issued
Authors and funding
6 authors.
Funding
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.
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