The basic steps of genome editing in bacteria is the introduction of exogenous template DNA in the form of plasmids, ssDNA or dsDNA and the subsequent integration into the chromosome via homologous recombination. However, large-size modifications, incompatibility of individual tools as well as high-throughput systems are still challenging. Novel technologies like CRISPR/Cas9 and large scale DNA synthesis brought genome editing within reach of a broad scientific community. In recent years, the relevance of genome editing in bacteria rapidly increased in basic research, biotechnology and synthetic biology. Its multi-color scarless co-selection system significantly improves editing efficiency and provides visual quality controls throughout the assembly and editing process. The modular character facilitates DNA library applications, and recycling of standardized parts. CRISPR SWAPnDROP also provides common genome editing approaches comprising scarless, marker-free, iterative and parallel insertions and deletions. dadantii without size-limiting intermediate DNA extraction. We demonstrate the excision, transfer and integration of large chromosomal regions between E. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli, the fast growing Vibrio natriegens and the plant pathogen Dickeya dadantii. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. CRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species.
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