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In Jinek et al., the authors show nuclease activity of their CRISPR/Cas9 system using the so-called Surveyor assay method. This assay recognizes small mismatches in dsDNA which are introduced by error prone non-homologuous end-joining after dsDNA breaks have been introduced by some foreign agent (such as Cas9). The Surveyor assay then -again- breaks the DNA at those points where it recognizes mismatches. The newly broken DNA fragments can be assayed and so one can identify the position of the initial, Cas9 dependent breakage.

My question is: for direct demonstration of Cas9 activity at the programmed position, why not first silence non-homolguous end-joining repair mechanisms in the cell and then directly analyse the lysate? Is this technically not feasible? Or is it not done in order to not introduce another change in the cell for which there would be no control?

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  • $\begingroup$ Because Double Stranded breaks will initiate apoptosis if they are not repaired, so you can't really tell if it is an effective method of gene editing if the cells die. Also you would need to see how many cells survive to determine transfection and site directed recombination efficiency. You also likely want to see how the breaks resolve in this experiment so silencing NHEJ would not give you that information. $\endgroup$ – AMR Sep 20 '15 at 16:56
  • $\begingroup$ @AMR You make good point about apoptosis. I'm not sure, but are these cells dividing after having been transfected? I dont understand what you mean by your last sentence. $\endgroup$ – TMOTTM Sep 22 '15 at 18:01
  • $\begingroup$ Cells are dividing after transfection. A bit too long for comments but read Materials and Methods either from that paper or any of the other related papers on CRISPR Cas9. The first generation of engineered Cas9 nucleases were double strand cutters, so breaks are resolved via Non Homologous End Joining, as you pointed out. NHEJ introduces random nucleotide insertions, so to see how these were resolved. In other words you are looking at how the cell would behave if, say, you wanted to use this for site directed knock outs. You want the data that comes from the repair itself. $\endgroup$ – AMR Sep 22 '15 at 19:53

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