Team:Bulgaria/second-project
Second project
Optogenetic CRISPR mediated mutagenesis
Most in vivo mutagenesis methods rely on the use of toxic chemical mutagens or hazardous radiation to increase the mutation rates. The nature of these agents limits their use as part of the educational process since they present a serious danger for a beginner with no lab experience. There are safer alternatives like transposon-active strains or mutator strains but they lack precise control and can cause unnecessary mutations.
Our aim was to design a new approach for in vivo mutagenesis that is safe for beginners, efficient and easy to control. We decided to use optogenetic production of ROS to damage DNA and introduce mutations in E. coli. Our selected tools to induce ROS production in vivo are three genetically encoded photosensitizers - KillerOrange, SuperNova, and mini singlet oxygen generator (miniSOG). All of them need only light in the visible part of the spectrum to induce ROS production and therefore they can be considered as safe options for students with no or low lab experience. Moreover, the level of produced ROS can be easily controlled via the intensity of the used light and/or the time of exposure.
We used two specially designed LED-based devices to illuminate our bacteria. In order to further improve our system we decided to fuse the three ROS generating proteins to dCas9. dCas9 will serve as a guiding platform concentrating the ROS production in selected regions of interest We expect that this will increase the number of mutations in given genomic areas.
To test if CRISPR-dCas9 can serve as targeting platform for optogenetic ROS generators we decided to use the following proteins - KillerOrange, SuperNova and Mini Singlet Oxigen Generator (MiniSOG). A SuperNova construct was avialable at the iGEM registry (BBa_K1491017). The other two CDSs were synthesized as gBlocks by IDT, cloned in pSB1c3 and submitted to the registry by iGEM Bulgaria.
From all three proteins only MiniSOG was used before as a mutagenesis tool in C. elegans ("Optogenetic mutagenesis in Caenorhabditis elegans", doi:10.1038/ncomms9868). In this work MiniSOG was fused to a histone. This fusion protein induced heritable mutations in a light-dependent manner thanks to the ROS production in close proximity to the DNA molecules.
We tried to expand this conception and make it applicable in E. coli. First we increase the number of optogenetic ROS generators used and codon optimized them for bacterial host. Next we decided to fuse them to a dCas9 protein. In this way one can target these fusion proteins to given genomic regions of interest. To fuse the protein we used a short and flexible protein linker between the dCas9 and our three ROS generators. Having in mind the large dCas9 sequence that contains target sites for most of the BioBrick restriction enzymes, we needed a new sequence and ligation independent cloning method. We decided to use the Aqua cloning approach. Our transformations obtained large number of colonies for all three constructs. Unfortunately, we were able to verify via colony PCR only one protein fusion - dCas9-KillerOrange. We plan to try different cloning strategies (SLIC and CPEC) to check if we will be able to generate the remaining two fusions.
To test our conception we cloned two gRNAs for the gene oppA in our gRNA expression vector (using Amp resistance backbone this time). These constructs were tested via transformation in bacteria with cas9 plasmid (no colonies were obtained for both constructs). The oppA gene encodes a periplasmic oligopeptide-binding protein in E. coli. There is a relationship between spontaneous aminoglycoside resistance in Escherichia coli and a decrease in oligopeptide binding protein (J. Bacteriol. October 1998 vol. 180 no. 20 5484-5488). We plan to use this in our proof-of-principle experiment. In case ROS mutagenesis increases the mutation levels in oppA upon light irradiation, we do expect a significant increase in the number of sporadic kanamycin resistant mutants.
We have a LED light device to treat our cells. It was built upon request by our friends from Sofia Technical University. Unfortunately, due to the cloning difficulties and other time limitations, we were not able to complete these experiments in time for the Wiki freeze. We hope to present some results at the Giant Jamboree on our talk and poster.
