The marine bacterium Vibrio natriegens is the fastest-growing non-pathogenic bacterium known to date and is gaining more and more attention as an alternative chassis organism to Escherichia coli. A recent wave of synthetic biology efforts has focused on the establishment of molecular biology tools in this fascinating organism, now enabling exciting applicationsfrom speeding up our everyday laboratory routines to increasing the pace of biotechnological production cycles. In this review, we seek to give a broad overview on the literature on V. natriegens, spanning all the way from its initial isolation to its latest applications. We discuss its natural ecological niche and interactions with other organisms, unveil some of its extraordinary traits, review its genomic organization and give insight into its diverse metabolismkey physiological insights required to further develop this organism into a synthetic biology chassis. By providing a comprehensive overview on the established genetic tools, methods and applications we highlight the current possibilities of this organism, but also identify some of the gaps that could drive future lines of research, hopefully stimulating the growth of the V. natriegens research community.

CRISPR/Cas9 is a powerful tool for genome editing. We constructed an easy-to-handle expression vector for application in the model organism Phaeodactylum tricornutum and tested its capabilities in order to apply CRISPR/Cas9 technology for our purpose. In our experiments, we targeted two different genes, screened for mutations and analyzed mutated diatoms in a three-step process. In the end, we identified cells, showing either monoallelic or homo-biallelic targeted mutations. Thus, we confirm that application of the CRISPR/Cas9 system for P. tricornutum is very promising, although, as discussed, overlooked pitfalls have to be considered.

The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for fundamental research and biotechnology. To fully harness the potential of this bacterium, highly efficient genome editing methods are indispensable to create strains tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This highly efficient natural transformation is able to mediate uptake of multiple DNA fragments, thereby allowing for multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR-Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR-Cas9 targeting the wild type sequence, and thus leading to death of non-edited cells. Through cell killing with efficiencies of up to 99.999%, integration of antibiotic resistance markers became dispensable, enabling scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100%. Further, we confirmed its applicability for simultaneous deletion of multiple chromosomal regions. Lastly, we showed that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby broadens the target spectrum.