Why
In our experiment, the production of differentially identifiable expression patterns is paramount. A library of promoters with robust expression patterns was generated to achieve this (step 3 to 5). To further modulate the expression patterns, a promoter targeted short guide RNA (sgRNA)-dcas9 repression system was devised. Using such a system will add a third expression level to the already existing ON and OFF states of our system, expanding the options further.
The sgRNAs have a 20bp ‘SEED’ region homologous to the promoters (used in step 3) between the -10 and -35 box, which will form a complex with a deactivated Cas9. The complex will be directed to the promoter through the homology between promoter sequence and the sgRNA. Binding of this very big RNA-protein complex to the promoter sequence will inhibit recognition and binding of RNA polymerase to the promoter region due to allosteric hindrance. The strength of inhibition is related to the binding affinity of the complex to the target DNA. Ideally, each promoter will have its own unique sgRNA which only inhibits the intended promoter to prevent cross interference.
How
To exemplify that the presence of a repression system would allow an increase in possible key outputs so we decided to start with a simple proof of function experiment. Therefore, we decided to go for a two plasmid based system.
One low copy vector was used to express both the catalytically inactive Cas9 protein and the RFP reporter, fused to any of the five promoters previously tested in step 3. Using a low-copy plasmid for the expression of reporter and dCas9 was a precautionary measure, as high levels of dCas9 can be toxic to the cells due to unspecific binding to other regions of the genome. Starting the experiments with only one reporter would allow us to easily establish how well each sgRNA represses the complementary promoter. The second plasmid was comprised of the sgRNAs and a high copy origin of replication (ori). Confining the sgRNAs to a separate vector is advantageous in that it can be easily substituted out for another vector, potentially detailing sgRNAs with a differing affinity to the promoter. This interchangeability can facilitate a ‘plug and play’ system to produce a multitude of expression levels with ease.
To select for a dual transformation, each vector backbone requires separate antibiotic resistances. Consequently, the high copy vector (containing sgRNAs) was given an chloramphenicol resistant cassette, the low copy backbone was given a erythromycin resistance cassette.
As a control, a sgRNA 0 was added to the library. The ‘SEED’ region of this sgRNA is not homologous to any of the promoters. This will act as a control, to signify how the expression will differ when inhibition is occurring. It will also serve later to increase the pool of possible expression patterns when we come to the random ligation experiments . These were cloned using traditional type II restriction cloning with BsaI sites.
To construct the sgRNAs, primer dimers were PCR amplified to generate the initial fragments of the sgRNA, it’s promoter, and terminator; also with their complimentary BsaI sites. After digestion with BsaI, the fragments were linearly ligated to produce promoter-sgRNA-terminator ‘bricks’. These were then amplified using a set of ‘universal’ primers designed to be capable of amplifying all of our ‘bricks’, these primers were complementary to a multiple cloning site(MCS) flanking each brick we produced. These sgRNA bricks were then digested using this MCS and ligated into their associated high copy backbone.
Results
The above graph shows characterization of the sgRNAs. As you can see, gRNA3, 4 and 5 are repressing well. gRNA 1 and 2 have no repression, and PE shows that there is very little expression as a background level.
