2025/06/19 by Sebastian Gude, Andreas Birk Bertelsen, Morten H. H. Nørholm · 1 voice
Biochemistry, Genetics and Molecular Biology · #Bacterial Genetics and Biotechnology #CRISPR and Genetic Engineering #RNA and protein synthesis mechanisms
paper · doi:10.1101/2025.06.19.660532
openalex publication_date 2025/06/19 · openalex created_date 2025/06/21 · openalex updated_date 2026/07/14
Abstract We have developed an easy-to-use in vivo method to iteratively relocate functional chromosomal loci onto an episome in Escherichia coli by utilizing synthetic DNA fragments. In this in vivo cut’n’paste procedure, “cutting” is executed by the RNA-guided DNA endonuclease Cas9 and a set of guides, while “pasting” is facilitated by the phage λ Red recombinase, which are all synthesized on easily curable helper plasmids. To demonstrate the utility of in vivo cut’n’paste, we commercially obtained synthetic DNA fragments containing locus-specific homology regions, antibiotic marker cassettes, and standardized Cas9 target sequences, and successfully relocated seven functional chromosomal loci. Scarless relocation mutants of the trg , aer , tsr , malHM , malQT , macB - nadA , and folA chromosomal loci were obtained as antibiotic-resistant isolates by combining Cas9 counterselection with the restoration of an antibiotic marker cassette. The additional antibiotic marker cassettes and standardized Cas9 target sequences present in the synthetic DNA fragments are inherently eliminated upon completion of the procedure, enabling iterative processing of chromosomal loci. In vivo cut’n’paste should be widely useful, especially in genome (re-)engineering efforts of E. coli and other bacteria because the procedure can be performed by non-specialists in unmodified wild-type cells. Significance Editing of genomes is crucial to understand the inner workings of bacteria and to alter them for biotechnological applications. In vivo cut’n’paste, in analogy to the frequently used keyboard shortcuts Ctrl-X and Ctrl-V which allow to move text, images, or files on computers, makes it easy to relocate large chunks of the bacterial genome without requiring skills to manipulate DNA outside of the bacterial cell. Like Ctrl-X and Ctrl-V, in vivo cut’n’paste can be executed several times to iteratively edit multiple genomic locations in the same bacterium. Since in vivo cut’n’paste entirely takes place inside the bacterial cell, it is particularly well suited to edit hard-to-tackle genomic features such as essential genes.