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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - dCas9 (Dissection of the mammalian transcription termination mechanism by CRISPRi technology.)

Teaser

Summary

Proteins in the cell are made according to the mRNA template, synthesised by DNA-dependent RNA polymerase according to the DNA template in what is called transcription process. However the mRNA template is suitable for protein synthesis only if RNA polymerase starts and stops this synthesis, or transcription, at very specific points.
While DNA of the â€œsimplerâ€ organisms, such as bacteria, contains well-defined nucleotide sequence signals, recognised by polymerase as start and stop signs, principles of human DNA â€œpunctuationâ€ are less clear.
One of the proposed stop mechanisms in human genes depends on polymerase slowdown. In the dCAS9 project we directly tested if such a slowdown can work as a stop trigger for genes in the cells. To check this hypothesis, we employed a modified bacterial dCas9 protein, which binds to a specific nucleotide sequence on the DNA. This protein was positioned on the way of transcribing polymerase as a sort of roadblock. We demonstrated that such an obstacle is sufficient to induce polymerase slowdown and eventually transcription termination in a way similar to native pause-induced termination type. Importantly, this happens only after polymerase passes certain â€œend of mRNAâ€ signal. We also demonstrated, that this dCAS9 system can to some extent rescue genes in cases when termination is altered by external factors, such as osmotic stress. This work allowed to check the influence of an isolated factor on termination process for the first time and expands our knowledge about its mechanism.

Work performed

While working on this project, we selected a number of the experimental genes based on the data developed in the lab in the previous years. We developed appropriate system for delivery of dCas9 protein and guiding RNAs (sgRNA) into the cells, combining previously published approaches and some in house improvements. We validated the efficiency of our optimized system for the previously described dCas9 repression targets. On the next step we generated guiding RNAs for selected experimental genes to target each gene with several dCas9 molecules. We characterised the effect of Cas9 block for a set of genes, and also followed changes in termination osmotic stress resistance. Experimental results have been presented on two international conferences so far, and will be put together into publication manuscript to reach wider scientific community.

Final results

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 â€œend of mRNAâ€ 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 â€œend of mRNAâ€ 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.