ArticleNucleic acids research2021
A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy.
Article in Nucleic acids research, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Epigenetic regulation in prokaryotes: transcriptional and phenotypic outcomes of DNA methyltransferase activity.FEMS microbiology reviews · 2026Review
- Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli.Biotechnology letters · 2025Article
- Advances in Diversity, Evolutionary Dynamics and Biotechnological Potential of Restriction-Modification Systems.Microorganisms · 2025Review
- Efflux Pumps and Porins Enhance Bacterial Tolerance to Phenolic Compounds by Inhibiting Hydroxyl Radical Generation.Microorganisms · 2025Article
- Adaptive Evolution for the Efficient Production of High-Quality d-Lactic Acid Using EngineeredMicroorganisms · 2024Article
- CRISPR-Cas9-assisted genome editing inbioRxiv : the preprint server for biology · 2024Article
- Microbial Cell Factories in the Bioeconomy Era: From Discovery to Creation.Biodesign research · 2024Review
- Construction and functional verification of size-reduced plasmids based on TMP resistance geneMicrobiology spectrum · 2023Article
- Using a synthetic machinery to improve carbon yield with acetylphosphate as the core.Nature communications · 2023Article
- Construction of a bacteriophage-derived recombinase system in Bacillus licheniformis for gene deletion.AMB Express · 2023Article
- Synthetic biology tools for engineeringComputational and structural biotechnology journal · 2023Review
- Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity.Microbial cell factories · 2022Article
- Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.Microbial cell factories · 2022Article
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Authors and funding
9 authors.
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Abstract
The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.
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