Generalized bacterial genome editing using mobile group II introns and Cre‐lox

• Published in: Molecular systems biology Vol. 9; no. 1; pp. 685 - n/a
• Main Authors: Enyeart, Peter J, Chirieleison, Steven M, Dao, Mai N, Perutka, Jiri, Quandt, Erik M, Yao, Jun, Whitt, Jacob T, Keatinge‐Clay, Adrian T, Lambowitz, Alan M, Ellington, Andrew D
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 2013; EMBO Press; Nature Publishing Group; Springer Nature
• Subjects: Bacillus subtilis; Bacillus subtilis - genetics; Bacteria; bacterial genome engineering; Base Sequence; Cre‐lox; Deoxyribonucleic acid; DNA; E coli; Editing; Efficiency; Engineering; Escherichia coli; Escherichia coli - genetics; Genetic engineering; Genetic Engineering - methods; Genetic Loci; Genome editing; Genome, Bacterial; Genomes; Insertion; Integrases - genetics; Integrases - metabolism; Introns; Inversions; Lac Operon; LacZ gene; Liquid oxygen; Loci; mobile group II introns; Molecular Sequence Data; Mutagenesis, Insertional; Nucleic Acid Conformation; Pastes; Plasmids; Polyketide synthase; Proteins; Recombination, Genetic; Ribonucleic acid; RNA; Scholarships & fellowships; Sequence Deletion; Sequence Inversion; Shewanella - genetics; Shewanella oneidensis; Staphylococcus aureus; Staphylococcus aureus - genetics; United States > US; Ohio
• ISSN: 1744-4292; 1744-4292
• DOI: 10.1038/msb.2013.41

Efficient bacterial genetic engineering approaches with broad‐host applicability are rare. We combine two systems, mobile group II introns (‘targetrons’) and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one‐step cut‐and‐paste operations. We demonstrate insertion of a 12‐kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one‐step cut‐and‐pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre‐mediated manipulations typically approaches 100%. A general bacterial genome engineering framework, ‘Genome Editing via Targetrons and Recombinases’ (GETR), is presented. GETR combines mobile group II introns (targetrons) and the Cre/lox system to allow genomic manipulations at a large scale. Synopsis A general bacterial genome engineering framework, ‘Genome Editing via Targetrons and Recombinases’ (GETR), is presented. GETR combines mobile group II introns (targetrons) and the Cre/lox system to allow genomic manipulations at a large scale. The combination of targetrons and Cre/lox represents a broad‐host range solution to genome editing. Engineered targetrons were used to deliver lox sites site‐specifically into the bacterial genome. Targetrons carrying lox sites were used to generate large‐scale insertions, deletions, inversions, and unique cut‐and‐paste operations in bacterial genomes.