When repair guiding DNA is transfected into cells, how does it reach the nucleus to aid in the repair of DNA breaks?
On a side note, say I transfect cells with Cas9 mRNA and template DNA, would this alone translocate Cas9 to the nucleus? I would presume not, how could one accomplish this?
Kind regards,
Jelle Bonthuis
Edit 1: I understand that it is possible to use a nuclear signalling sequence on the sgRNA to reach the nucleus, my question is primarily regarding the expressed Cas9 protein and the template DNA.
That do you mean DNA template? How do you transfect your cells? I have never heard about specific sequense in RNA for nucleus delivery. You need nuclear localization signal for protein, but not for RNA. The same thing with DNA. If you try to transfect your cells with plasmid it will be still delivered to the nucleus because specific nucleafection agent as lipofectamine or turbofect, as well as by making holes in into the cell if you use electroporation. Precise mechanism of nucleic acid into nucleus is still unknown.
Thanks maybe I should ask a more clear question:
Say I would want to genetically modify an animal in vivo. If I would use lipid carriers for RNA and DNA, at least some cells would be transfected. How would the cas9 protein translucate to the nucleus (after being transcribed by the mRNA provided), how would the sgRNA reach the nucleus and how would the template DNA reach the nucleus. If this does not happen on its own, what would be needed to do this. Could lipofectamine be used in the blood stream?
If you use nucleofection for DNA transfection, it will be delivered to the nucleus.
Nucleofection is electroporation with specifc buffers and specific conditions.
Some of the DNA will also be delivered to the nucleus if you use liposomes for transfection.
In Eukaryotes, transcription takes place in the nucleus and then RNAs are exported to the cytoplasm where translation takes place.
Jelle, unfortunately, no one can gives you the answer how delivery of nucleic acid to the nucleus work. There is only theories which have no proofs. But there are tools which work for sure- transfection and electroporation. If it will help you I attached new publication about in vivo genome editing without viruses.
If you use both gRNA and Cas9 plasmids or chemically synthesized gRNA or mRNA of Cas9 they will be delivered to the cell with one of the methods I mentioned before. But I think you should check if your plasmid or mRNA contain nuclear localization signal in sequence of Cas9 (for gRNA it isno necessarly). Everything else will be done by the cell.
Here people use lipofectamine 3000 for in vivo transfection-https://www.researchgate.net/post/What_is_the_best_way_of_delivering_siRNA_in_vivo
I should notice, that high effciency of transfection you will observe in liver. But muscle, for example, will be hard to transfect.
This article might be worth a read for you:
Article Nuclear entry of nonviral vectors
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