I am aware that there are two different methods for double strand break repair (non-homologous end joining or homology directed repair), but what I wanted to know is if the repair mechanism restores the same number of nucleotides which used to be there. So if I use the Cas-9 enzyme to excise a certain 20-nucleotide sequence, will the repair mechanism bring back 20 nucleotides as well?
If you let your Cas9 cut at two different spots 20 base pairs apart your organism will probably just attach the ends together with non-homelogous end joining, unless a homologic region is present. So there will be plenty of cases in which you get a clean 20 nuclotide exicision from your sequence, though there might be some cases where there will be some random nuclotides added in between, so I would sequence your sample, to make sure what you got. Your organism doesn't know what you will cut out, so getting exaclty 20 nucleotides in will be unlikely (unless the piece you just cut out gets put back in, but that Cas9 will just cut again)
I would however take a look to see if Cas9 can cut two pieces so close to each other, I think that I remember that there is a minimum distance between two cas9 sites to make it efficient, thought this might be something like 7 base pairs. I would double to check to make sure ;)
NO, Cas9 creates a double stranded break in the DNA. The DNA is generally repaired by non-homologous end joining which results in small deletions of 2-30 nucleotides. This cannot be controlled very precisely. However, if you include a template for repair, a long oligo or piece of DNA, then the break can be repaired by homologous recombination in which the added DNA is used as a template for repair.
Thanks for the responses! If I would like to use CRISPR to delete a certain segment of DNA, should I just let the system repair itself by non homologous end joining? And if so, are you saying that there will be a few cases where there are a few extra nucelotides added or deleted?
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