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inblue01 t1_iy0cn7w wrote

Some good answers here, but one piece is missing : screening!

You are correct: in most cases, the cells will repair the genome in a way that does not suit your downstream applications. It's a matter of probability : one in many cells will, by chance, repair your DNA with your template, or do the desired mutation.

One step that you need to do is to isolate clones (single cells), let them grow to a decent population and then screening (by sequencing and functional testing) these clones to determine which one has the desired insertion/mutation.

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fertthrowaway t1_iy1s8at wrote

With repair template and active enough homologous recombination, you can get like 99-100% correct edit efficiency in many organisms.

If you rely on NHEJ after the nuclease cut, then you basically have to screen through repaired DNA craters though, yeah.

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WiwaxiaS t1_iy202zz wrote

Ah yes, that's why indicator genes may be co-introduced, like GFP for instance, and sure functional testing for the gene products themselves can also be done.

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