ArticleNucleic acids research2017
Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases.
Article in Nucleic acids research, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed, 50 citations in OpenAlex.
- Silica Nanoparticles Block Natural Genetic Transformation inACS omega · 2025Article
- Article
- Improved DNA binding to a type IV minor pilin increases natural transformation.Nucleic acids research · 2025Article
- DNA repair is essential forJournal of bacteriology · 2025Article
- SbcB facilitates natural transformation inJournal of bacteriology · 2025Article
- DNA repair is essential forbioRxiv : the preprint server for biology · 2025Article
- Microbial cross-feeding stabilized by segregation of a dependent mutant from its independent ancestor.The ISME journal · 2025Article
- Cyclic Diguanylate in the Wild: Roles During Plant and Animal Colonization.Annual review of microbiology · 2024Review
- DNA binding is rate-limiting for natural transformation.bioRxiv : the preprint server for biology · 2024Article
- Expanding the capabilities of MuGENT for large-scale genetic engineering of the fastest-replicating species,Microbiology spectrum · 2024Article
- Efficient natural plasmid transformation ofPNAS nexus · 2024Article
- Microbial Cell Factories in the Bioeconomy Era: From Discovery to Creation.Biodesign research · 2024Review
- Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.Nucleic acids research · 2023Article
- Expanding natural transformation to improve beneficial lactic acid bacteria.FEMS microbiology reviews · 2022Article
- A multifunctional system for genome editing and large-scale interspecies gene transfer.Nature communications · 2022Article
- Vibrio cholerae requires oxidative respiration through the bd-I and cbb3 oxidases for intestinal proliferation.PLoS pathogens · 2022Article
- Motor-independent retraction of type IV pili is governed by an inherent property of the pilus filament.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Fresh Extension of Vibrio cholerae Competence Type IV Pili Predisposes Them for Motor-Independent Retraction.Applied and environmental microbiology · 2021Article
- Natural Transformation ofFrontiers in microbiology · 2021Article
- Article
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
Abstract
Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
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Registered trials
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