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-Metacelsus- t1_iy0cbl2 wrote

I do this regularly as part of my research. Here's how it works:

I make a bacterial plasmid that contains the DNA that I want to insert. On either side of this DNA, I have an additional 1000 bases of DNA that has the same sequence as my target site. These are known as homology regions. I can assemble this plasmid using a method such as Gibson assembly.

I then introduce this plasmid into the cells, along with another plasmid expressing Cas9 and guide RNA, using electroporation. The Cas9 and guide RNA cut the target site. The cell then tries to repair it.

The usual repair pathway is called non-homologous end joining, which simply sticks the DNA back together. This is not what I want. However, cells can also repair DNA through homology-directed repair (HDR), where they basically look for similar sequences and swap them into the cut site.

When cells perform HDR, they can use my plasmid to perform the repair because it has the homology regions. Once this happens, the DNA sequence becomes inserted into the target site.

For a good intro-level review of this, I recommend: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5901406/

(Note that there are other ways being developed to do edits with CRISPR, I'm just explaining HDR)

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pitchapatent t1_iy0kkcz wrote

Wonderful reply! I would just emphasize/clarify that the HDR template (the DNA that you want to paste) doesn't actually get incorporated into the cell's genome. It merely serves as a guide, allowing the cell's repair machinery to copy the sequence info into the genome. I know you know this, but it's one of the most counterintuitive and easy-to-miss elements of the pathway.

For another entry-level resource on CRISPR everything, I would recommend CRISPRpedia - I've linked the "technology" page and it has an HDR section. But it also glosses over the it's-not-actually-pasted-in aspect that I addressed above. Even worse, this video shows something at 3:00 that simply does not happen (repair template being physically pasted into the genome).

This video does show an accurate depiction of the repair mechanism, with the good stuff starting at 1:00. Although this is not a CRISPR-specific video, the mechanism is very similar. Just think of the pink DNA as the repair template - the scientist-provided sequence-to-be-pasted. This is the single best resource I'm aware of that succinctly addresses the question posed by /u/AutomaticAd1918

OP, since you asked about how these things get into cells, you should check out the "Delivering CRISPR therapies" section of this CRISPRpedia page on genetic medicines. In brief, scientists can use viral vectors (widely used in gene therapy), lipid nanoparticles (same tech as COVID vaccines), or physical/mechanical means to get large molecules into cells. Although that page focuses on getting the CRISPR enzyme into a cell, the same approaches work for delivery of the HDR repair template (the DNA to be pasted). Delivery is a major challenge because cell membranes are fiercely dedicated to acting as a barrier, and they're very effective at preventing transit of larger molecules. Traditional small molecule drugs don't face the same delivery challenges because they can "slip through the cracks" and enter cells via diffusion.

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trijammer t1_iy2l7md wrote

Thanks for this clarification. The video of the repair process is great.

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HappyAntonym t1_iy0ruo3 wrote

Woah, great explanation! It sounds like you're basically creating a Trojan Horse out of DNA.

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TheDurrrmanNeighbor t1_iy11k58 wrote

I remember back in the day there was an episode of a cartoon where they tried to pull off a bank heist. The character spent time devising a plot and the simplest plan without fail was to create path.

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[deleted] t1_iy2gm7i wrote

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-Metacelsus- t1_iy34e5r wrote

> What I don’t understand is how this would work for an entire body?

Your understanding is correct, because it doesn't work for an entire body, the efficiency per cell is not nearly good enough. If you want a full-body edit you would have to edit stem cells, select the edited cells you want, and then use various embryology techniques to put the stem cells in an embryo and have them develop into a new organism.

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[deleted] t1_iy34o2b wrote

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CrateDane t1_iy50gdd wrote

Depends on the gene defect; if it's enough to edit 3% of your liver cells to produce an important protein to circulate in your blood, then it's pretty reasonable to expect to cure that disease with (non-germline) CRISPR/Cas.

If it's something that needs to be fixed in 100% of a certain cell type, especially non-proliferating cells, then that's going to be very tough. And if it's something that acts during development, then fixing the DNA in an adult would do nothing (the body has already been "built" with the wrong "blueprint").

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Astavri t1_iy16nnp wrote

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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