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In this paper1, I read that mutant versions of Cas proteins such as a deactivated Cas9 (dCas9) are used alongside a guide-RNA (gRNA) to form variants of CRISPR tool that can function as transcription regulators.

Since the mutant Cas9 has lost its nuclease functionality, the mutant gRNA-dCas9 complex can "block" transcription factors such as RNA polymerase from binding a certain gene, hence repressing transcription or elongation.

Another way that dCas9 can regulate transcription is by "carrying" another transcription factor - be it an activator or repressor - and this can be done by engineering a dCas9 fused to the other transcription factor of choice.

I understand how the dCas9 can block the binding of another transcription factor to repress transcription initiation or elongation, but what is the point of fusing another transcription factor with the dCas9? It seems to me that in this scenario, the dCas9 is just a passive carrier of the transcription factor that is actually doing the job, so why can't the transcription factor be fused with the gRNA instead, so that it can directly affect transcription when the gRNA forms complimentary base pairs with the target gene, instead of it being on a passive dCas9?

1. Jusiak, B., Cleto, S., Perez-Pinera, P. and Lu, T.K., 2016. Engineering synthetic gene circuits in living cells with CRISPR technology. Trends in biotechnology, 34(7), pp.535-547.

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so why can't the transcription factor be fused with the gRNA instead, so that it can directly affect transcription when the gRNA forms complimentary base pairs with the target gene, instead of it being on a passive dCas9?

The main reason for that is that both the gRNA and the dCas9-TF (transcription factor) combination can (each) be encoded genetically, while a combination of a gRNA with a transcription factor could only be generated chemically.

The possibility to genetically encode the dCas9-TF + gRNA unit allows one to:
a) use established methods for transferring DNA into cells,so that the cells make the Cas9 and the gRNA by themselves and
b) generate cells that will stably express the whole unit (dCas9-TF + gRNA) forever.

Neither of these options would be possible with a chemically linked gRNA-TF (which is also much harder to make for molecular biology labs that don't have the proper chemistry equipment).

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  • $\begingroup$ Thank you. So in a more general scenario, is it more conventional to induce the synthesis of a gRNA-Cas9 complex by the cell itself, rather than deliver the already-synthesized complex into the cell? $\endgroup$ – P. SN Aug 8 '18 at 9:15
  • $\begingroup$ I don't really have experience with Crispr/Cas9 myself, so I'm can't say for sure, but its is definitely easier to only transfer DNA (plasmids) in cells and let them build the proteins & RNA, since DNA transfection is one of the most common molecular biology methods. Transfection with protein &/ RNA is harder and therefore more unconventional (but still sometimes used for Cas9, especially if you DON'T want the cell to retain the Cas9 protein in any way) $\endgroup$ – Nicolai Aug 8 '18 at 11:43

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