Dataset Information

Efficient dual-negative selection for bacterial genome editing.

ABSTRACT: BACKGROUND:Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering. RESULTS:Here, we established a time-effective variant of consecutive single-crossovers. This method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in-frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Adapted versions enabled efficient gene editing also in Pseudomonas aeruginosa and multi-drug resistant (MDR) Escherichia coli clinical isolates. CONCLUSIONS:Our method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement.

SUBMITTER: Cianfanelli FR

PROVIDER: S-EPMC7245781 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Efficient dual-negative selection for bacterial genome editing.

Cianfanelli Francesca Romana FR Cunrath Olivier O Bumann Dirk D

BMC microbiology 20200524 1

<h4>Background</h4>Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering.<h4>Results</h4>Here, we established a time-effective variant of consecutive single-crossovers. This method exploits rapid plasmid construction using Gibson assembly, a conve ...[more]

PMID: 32448155

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Project description:BackgroundRice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria. Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants.ResultsWe introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants.ConclusionWe demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

Dataset Information

Efficient dual-negative selection for bacterial genome editing.

Publications

Efficient dual-negative selection for bacterial genome editing.

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