I want to knock out a gene using CRISPR, to design the arm do I need to replace the whole CDS by a marker gene (GFP) or just induce a frame shift mutation? please suggest.
Why even bothering with HDR when you just want a KO?
With HDR you can have a specific change in your CDS, either a frame shift by adding a base or an aminoacid exchange....
When you just want to make an KO, I think a normal KO approach would be sufficient.
When I'm completly wrong correct me.
Best regards.
The simple KO induced by double strand break it usually enough....
But some people saw that when making double strand breaks with nucleases, in some (rare) cases, the indels obtained were a multiple of 3, and so it did not induced a frame shift mutation, but just addition/deletion of some amino acids.
One thing you can do to prevent this, is using an oligo (modified or not) with several STOP codons in it, in all the frames, to improve to rate of mutation (you can introduce a restriction enzyme site in the oligo juste to check how the cells were KI-modified with the oligo). Maybe you can see this article, for the strategy of using ODN
http://www.cell.com/cell-reports/fulltext/S2211-1247%2816%2930104-8
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