Developing CRISPR-Cas-Based Genome Editing Tools for High-Throughput Engineering of Non-Model Prokaryotes

Although model organisms such as Escherichia coli and Saccharomyces cerevisiae have shown to be efficient industrial work horses for production of green chemicals and fuels from biomass, the use of alternative production hosts could provide important operational advantages to create more sustainable and economically feasible processes¹. However, the development of such non-model organisms into cell factories is hampered by a lack of knowledge on their metabolism and underdeveloped or absent genetic tools for the required metabolic engineering^1,2. Established methods for genomic modifications such as recombineering or plasmid-based homologous recombination (HR) have a high wild-type revertant rate, resulting in low engineering efficiencies, thus requiring extensive screening or insertion of markers. This also limits the use of these methods in organisms with low transformation and recombination efficiencies. Contrary to eukaryotes, most prokaryotes do not have a functional non-homologous end-joining system and double-strand DNA breaks (DSBs) induced by Cas9 (or other Cas-like molecules) are lethal, which forms the basis for using CRISPR-Cas-systems in prokaryotes as powerful counter-selection system when combined with a recombination method³. This has strongly increased engineering efficiencies in E. coli as well as several non-model organisms^2-4. A major issue for non-model organisms is the lack of genetic parts such as plasmids, markers and tightly controllable promoters. Therefore, the Cas9-based system should be as simple as possible. Recently, a Streptococcus pyogenes (sp)Cas9-based method was developed in the facultative thermophile Bacillus smithii, based on a single plasmid containing HR-flanks, the gene coding for spCas9 and an sgRNA targeting the wild-type sequence selected for mutagenesis via HR⁴. The system does not need inducible promoters as it was found that spCas9 is inactive in vivo ≥42°C, and its activity can thus be controlled via the growth temperature⁴. This enabled highly efficient genome engineering by transformation and HR at elevated temperatures when spCas9 is inactive, and subsequent counter-selection of non-edited cells at 37°C by active spCas9⁴. Here, we apply this method in other non-model organisms to extend the range of bacteria for which this efficient Cas9-based counter-selection can be used. This also includes optimization or establishment of HR-methods. Altogether, by further developing the genome editing toolbox for these organisms we aim to broaden the range of potential cell factories to species that are for example more tolerant to low pH, high temperature and toxic substrates and products.

References

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3. Mougiakos, I.*, Bosma, E.F.*, de Vos, W.M., van Kranenburg, R., van der Oost, J., Next generation prokaryotic engineering: The CRISPR-Cas toolkit. Trends Biotechnol. 34, 575-87 (2016).
4. Mougiakos, I.*, Bosma, E. F.*, Weenink, K., Vossen, E., Goijvaerts, K., van der Oost, J., and van Kranenburg, R. Efficient genome editing of a facultative thermophile using the mesophilic spCas9. ACS Synth. Biol. 6, 849-861 (2017).