In the course of implementation of this project, we changed previously available dCas9/sgRNA (CRISPRi) systems to improve the stability of sgRNAs, delivery of dCas9 to the nuclei, and optimized other parameters. We validated the system efficiency for CRISPRi targeting and thus generated an improved protocol, which can be easily applied transcription termination studies. After validating the system, the genes were targeted in vivo and the changes caused by such a targeting were determined by comparison with mock-treated cells.
This, in this work we first in the field describe the application and efficiency of CRISPRi systems beyond activating and repressing gene expression. Instead of previously reported regulation of mRNA levels up or down, here we apply it for the studies of the basic mechanisms of nascent RNA generation.
We demonstrated, that bringing a pausing factor on the way of transcribing RNA polymerase indeed did cause termination, without changing the DNA sequence. We examined the effects of dCas9 positioning relative to “end of mRNA” signal on the efficiency of the termination shift. We discovered that the block can change termination profile only if it is put downstream of the “end of mRNA” signal, which is in line with previous data from different laboratories, implying that a functional signal is a prerequisite for termination.
Importantly, we demonstrated, that dCas9 alone is enough to shift termination area, and that additional modifications of DNA-binding proteins are not required for this process.
On the next stage of the project we followed the failure to terminate transcription induced by osmotic stress. We demonstrated, that though CRISPRi system does not restore original termination efficiency, it still acts as improving factor. Interestingly, the osmotic stress antitermination effect and CRISPRi anti-antitermination effects seem to be independent. This implies that these factors can be targeting not interrelated factors causing the termination. The exact factors are to be found during the follow-up time of this project.
Overall, as a part of the project we developed, fine-tuned and described a dCas9/sgRNA (CRISPRi) system, allowing to induce premature termination on the native cellular DNA in vivo. We examined the effects of such termination and resistance of resulting terminators to termination-blocking stress. The results from this work will be published once finalizing experiments will be completed. The results of this research are important for the transcription termination field and expand our understanding of the gene expression process.
