  The need for diverse chromosomal modifications in biotechnology, synthetic
biology and basic research requires the development of new technologies. With
CRISPR SWAPnDROP, we extend the limits of genome editing to large-scale in-vivo
DNA transfer between bacterial species. Its modular platform approach
facilitates species specific adaptation to confer genome editing in various
species. In this study, we show the implementation of the CRISPR SWAPnDROP
concept for the model organism Escherichia coli and the currently fastest
growing and biotechnologically relevant organism Vibrio natriegens. We
demonstrate the excision, transfer and integration of 151kb chromosomal DNA
between E. coli strains and from E. coli to V. natriegens without size-limiting
intermediate DNA extraction. With the transfer of the E. coli MG1655 wild type
lac operon, we establish a functional lactose and galactose degradation pathway
in V. natriegens to extend its biotechnological spectrum. We also transfer the
E. coli DH5alpha lac operon and make V. natriegens capable of
alpha-complementation - a step towards an ultra-fast cloning strain.
Furthermore, CRISPR SWAPnDROP is designed to be the swiss army knife of genome
engineering. Its spectrum of application comprises scarless, marker-free,
iterative and parallel insertions and deletions, genome rearrangements, as well
as gene transfer between strains and across species. The modular character
facilitates DNA library applications and the recycling of standardized parts.
Its novel multi-color scarless co-selection system significantly improves
editing efficiency to 92% for single edits and 83% for quadruple edits and
provides visual quality controls throughout the assembly and editing process.
