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FogeltheVogel t1_iy06eoh wrote

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

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

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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.

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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

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