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FogeltheVogel t1_iy01ut5 wrote

When DNA experiences a double stranded break (the type that CAS9 makes), there are 2 methods a cell has to repair it.

The first is the sloppy one, called non-Nomologous End Joining. The machinery for DNA repair can't really do anything with blunt breaks (the type that CAS9 makes), it needs ends that stick out a bit. Called literally Sticky Ends (if they overlap). Sticky vs blunt ends. So the first step is enzymes that remove some nucleotides from each end to make them sticky. After that, other enzymes come in that take these sticky ends and extend them into each other, repairing the break. The problem here is that some nucleotides get lost, and some random ones are added. This usually breaks the gene. When using CAS9 to knock out genes, this is sufficient.

The second method is what's used here. It's called Homologous Recombination, and it is not always possible to use. In essence, it uses the other chromosome as a template to accurately repair the DNA. Under normal conditions, this can't be used because the other chromosome is not readily available to serve as a template. During CRISPR treatments to insert a new geme, we provide a piece of DNA along with the CAS9 and it's guide RNA. This piece of DNA is the "new" gene, but it isn't incorporated like you originally thought. Instead, it is specifically designed so that the 2 ends of this piece of DNA are perfect matches to the 2 sides of the break made by CAS9. The DNA repair complexes will see thus use this as a template to 'repair' the break perfectly. Only in this case, they include extra nucleotides that were never part of the original strand.

I hope this helps.


CrateDane t1_iy060yz wrote

NHEJ can also be used for insertion, in strategies such as homology-independent targeted insertion (HITI). Because the ends being joined don't have to be homologous, you can co-deliver a linear DNA fragment and have the end joining pathway insert that where the double-stranded break was made.


FogeltheVogel t1_iy06eoh wrote

Is there any way to guide this process? It feels like this would have a rather low chance of actually happening.


Cleistheknees t1_iy0u6dt wrote

Preface: the last “i” in HITI is actually for “integration”, not “insertion” as stated above, in case you wanted to google around for more info.

You guide the process in the same way. The donor sequence used (theoretically used, anyways) in HITI is capped by the same sequences that gRNA-Cas9 is pointed at, and in fact is exceptionally accurate. The idea behind HITI is that it isn’t limited to actively differentiating cells.


deisle t1_iy24kmd wrote

You're right, all of these processes require that you have all the bits in the right place at the right time and the cell does the right thing and no enzyme messes up too hard. So you shove as much stuff in as you can to maximize your chances When I would try to insert a mutation in a zebrafish, I would inject hundreds of fertilized eggs at the single cell stage, let them grow up, and then take a tail clipping to genotype. I'd be lucky if I got a couple successful mutations from those hundreds of eggs. It's definitely a numbers game.

Caveat: this was like 6 years ago, when it was relatively new. Success rates have likely gone up as the technique has been refined but general principle remains


worotan t1_iy0hgwu wrote

That’s so clear for a complete laymen who is fascinated, thank you. It makes perfect sense.


Plantpong t1_iy0b0x7 wrote

Agreed with the above! NHEJ is often used for the insertion of 'random' nucleotides for generating gene knockouts. HR can more efficiently be used for targeted insertions when also introducing a repair template along with the CRISPR complex that has homology arms that match the target splice site.


gthing t1_iy1ljxx wrote

If my 23 and me shows me I have certain genes that are associated with higher risk for X,Y,Z - are those theoretically then curable with CAS9? Are the genes even understood enough to say if we switch one off it's not going to have some cascading weird effect or even that it will actually cure you? Last question: how long in your wild estimate until most everyday gene disorders are routinely cured during childhood?


wilnyb t1_iy27zw0 wrote

A major issue with correcting genes in adults is the the large number of cells that needs to be corrected. Let's say you have a muscle disease, hitting every single muscle cell in you body to correct an genetic disorder is very complicated. As of right now, this is easier with blood cells. You can isolate hematopoietic stem cells, that you can edit and then reintroduce in a patient. Those cell will repopulate the immune system.

Every disease is different. For many diseases it might be enough to correct 10% of the cells for a patient to be able to live with the disorder. Some of those examples already exist today. Some more complicated genetic disorders we might never be able to correct (at least in our life time).


HotDadBod1255 t1_iy36nd2 wrote

Unfortunately there really isn't much clinical data for in vivo gene editing, the only results so far are from two clinical trials from Intellia Therapeutics. Their results are really promising, but so far they've only shown they can do gene knockout in liver cells, which is pretty limited in scope. Hopefully them and some others are working on other ways to perform gene editing.