Background Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology. Results In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology. Conclusions The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.
IntroductionMicroorganisms colonize a wide range of natural and artificial environments. Even though most of them are unculturable in laboratory conditions, some ecosystems are ideal niches for bioprospecting extremophiles with unique properties. Up today, there are few reports concerning microbial communities found on solar panels, a widespread, artificial, extreme habitat. Microorganisms found in this habitat belong to drought-, heat- and radiation-adapted genera, including fungi, bacteria, and cyanobacteria.MethodsHere we isolated and identified several cyanobacteria from a solar panel. Then, some strains isolated were characterizated for their resistance to desiccation, UV-C exposition, and their growth on a range of temperature, pH, NaCl concentration or diverse carbon and nitrogen sources. Finally, gene transfer to these isolates was evaluated using several SEVA plasmids with different replicons to assess their potential in biotechnological applications.Results and discussionThis study presents the first identification and characterization of cultivable extremophile cyanobacteria from a solar panel in Valencia, Spain. The isolates are members of the genera Chroococcidiopsis, Leptolyngbya, Myxacorys, and Oculatella all genera with species commonly isolated from deserts and arid regions. Four of the isolates were selected, all of them Chroococcidiopsis, and characterized. Our results showed that all Chroococcidiopsis isolates chosen were resistant up to a year of desiccation, viable after exposition to high doses of UV-C, and capable of being transformed. Our findings revealed that a solar panel is a useful ecological niche in searching for extremophilic cyanobacteria to further study the desiccation and UV-tolerance mechanisms. We conclude that these cyanobacteria can be modified and exploited as candidates for biotechnological purposes, including astrobiology applications.
Background: Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology. Results: In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology. Conclusions: The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.
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