Abstract
The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.
Highlights
Second and third generation sequencing technologies, and in particular, shotgun metagenomics, have expanded our knowledge of the microbial diversity dramatically [1]
The landing pad was introduced into the chromosome via mariner-based transposon insertion, and the custom pathway integrated into the landing via Cre-lox recombinase-mediated cassette exchange (RMCE)
We aimed to develop a method that combines the universality of both CRISPR-Cas9 and chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the breadth of CRISPR-mediated editing to be applied as widely as Cre-lox functionality
Summary
Second and third generation sequencing technologies, and in particular, shotgun metagenomics, have expanded our knowledge of the microbial diversity dramatically [1]. The landing pad was introduced into the chromosome via mariner-based transposon insertion, and the custom pathway integrated into the landing via Cre-lox recombinase-mediated cassette exchange (RMCE). Since both the transposon and the Cre-lox recombineering systems were applied to diverse hosts, CRAGE enabled insertion of large biosynthetic gene clusters into 25 diverse gammaproteobacterial species. This approach does not provide capabilities beyond insertion, such as gene deletions or modulated gene expression
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