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Astavri t1_iy16nnp wrote

Reply to comment by -Metacelsus- in How exactly does CRISPR-CAS9 insert new genes? by AutomaticAd1918

You transfect the cas9 plasmid and not deliver the enzyme itself?

Do people not typically send the enzyme itself using electroporation? I was unaware of this part.

Basically all you are doing is a transduction/transfection then, with all the necessary genes being sent through.

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-Metacelsus- t1_iy19ucd wrote

Doing it with the cas9 plasmid is cheaper and works nearly as well. There is a greater chance of off-target edits though. But yes, if I was doing it clinically I would use the ribonucleoprotein.

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Astavri t1_iy1ba6r wrote

That's clever. I never thought about that as an option.

Do you have any publications or sources for doing it this way? If it's not any trouble.

I don't use crispr for anything at the moment, but there might be something I want to try it on. This 100% seems like the better option. And you could reuse the same plasmid with the cas9 and guide correct me if im wrong, and change the genes on the second plasmid if you want to try introducing a different gene in the same region?

Is it Ecoli or mammalian cells you are editing?

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CrateDane t1_iy4zwth wrote

You have multiple options. You can provide the Cas9 gene in DNA form, but also as mRNA. Or simply the protein, usually pre-assembled with guide RNA into a RNP.

Even the Cas9 DNA delivery has multiple options - viral vector, plasmid transfection, it all depends on the use case.

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