NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens.

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.

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens.

Vibrio natriegens is known as the world's fastest growing organism with a doubling time of less than 10 min. This incredible growth speed empowers V. natriegens as a chassis for synthetic and molecular biology, potentially replacing E. coli in many applications. While first genetic parts have been built and tested for V. natriegens, a comprehensive toolkit containing well-characterized and standardized parts did not exist. To close this gap, we created the Marburg Collection-a highly flexible Golden Gate cloning toolbox optimized for the emerging chassis organism V. natriegens, containing 191 genetic parts. The Marburg Collection overcomes the paradigm of plasmid construction-integrating inserts into a backbone-by enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V. natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organism-specific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V. natriegens community.

Vibrio natriegens: an ultrafast-growing marine bacterium as emerging synthetic biology chassis.

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 applications - from 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 metabolism - key 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.