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I've been reading up a bit on the CRISPR-Cas9 system for gene manipulation. From what I read, it introduces double-strand breaks at specific points determined by the choice of sgRNA. But how do you get from double-strand breaks to editing genes in incredibly specific ways? As far as I read you can fix point mutations, insert whole genes or disable genes with CRISPR-Cas9. How do you specify which kind of repair mechanism is invoked?

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But how do you get from double-strand breaks to editing genes in incredibly specific ways?

You should read the wikipedia page on CRISPR-Cas system. The target specificity is provided by the tracrRNA.

How do you specify which kind of repair mechanism is invoked?

The repair can be done either by Non Homologous End Joining (NHEJ) or Homologous Recombination (HR). If you provide excessive DNA template by transfection then it is likely to increase the chance of HR and this can be used for trangenesis.

When no template is provided then DNA can be repaired by HR with sister chromatin or NHEJ. Depending on the stage of the cell cycle one of these mechanisms is preferred over the other. HR is shown to be the most active in S-phase and starts declining in G2-phase.

So when you want to make transgenics or specific edits then co-transfect CRISPR-Cas with transgene DNA (with flanks homologous to target DNA sequence) or a homologous DNA with point mutation(s), preferably in cells at S-phase. It is possible to synchronize the cells using nocodazole or other molecules that arrest cell cycle.

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  • $\begingroup$ I assume "sgRNA", "tracrRNA", and "guide RNA" are all the same thing? And I can imagine getting efficient editing would be a problem, since you have to get at least 2 components into the cells, the Cas gene and guide RNA could be produced by 1 plasmid, but the template DNA to direct the repair would be separate, and you'd probably want several copies per cell to improve the odds of a successful repair. $\endgroup$ – user137 Feb 20 '15 at 15:07
  • $\begingroup$ @user137 yes pretty much the same thing.. It is not that difficult; transgenesis rates are much better than in plain HR. All these kind of techniques are followed by screening and selection. $\endgroup$ – WYSIWYG Feb 20 '15 at 17:19
  • $\begingroup$ My thinking comes from in vivo work, where you'd either have to package all the parts into 1 particle, or get get multiple molecules into the same cell. Though I've seen a paper where the corrected cells are accidentally selected for in vivo. $\endgroup$ – user137 Feb 20 '15 at 18:34
  • $\begingroup$ @user137 You transfect the ESC and then implant them in the ICM right? There is still scope for artificial selection. $\endgroup$ – WYSIWYG Feb 21 '15 at 6:43
  • $\begingroup$ @user137 If you want to learn more about the Cas9 system, I wrote a detailed explanation of the original publication by M. Jinek on my blog: qmviews.blogspot.ch/2015/09/… Would be glad to hear if it helped. $\endgroup$ – TMOTTM May 1 '16 at 13:16

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