I'm trying to insert few point mutations close to each other in a gene of interest and I want to use CRISPR/Cas9. Clearly, I am aiming at HDR rather than NHEJ, so I will provide a template DNA molecule that HDR can use to repair. There is a good number of PAM sites in my gene of interest, but since I need to target a single gene in a family of 3, I need to select the most specific region I can find. So, my question is: how close to the site I want to edit should the gRNA anneal? Is it important that the double strand break is inserted very close to the site I'm editing or it can be distant. If yes, how distant?
Hello
the closer you make the double strand break from your site of interest, the more efficient the modification will be.
I used a lot a designs of oligos (ssODN, dsODN, at different distances,...) maybe you can take a look:
Article Improved Genome Editing Efficiency and Flexibility Using Mod...
how long is the region you want to modify?
For example, with PS modified ssODN, you should be able to induce point mutations in a 100-150bp region with only one oligo (using around 25 homology arms on each side)
I need to change 3 bases that are within 9 nucleotides from each other, so very close. So you suggest to use a ssODN of around 100 bp, plus 25 bp homology arms at each side, is that correct?
i don't think you have to use such a long (100bp) ssODN. you can with only one PS modified oligo insert around 100-150bp.
For your 9bp distance, i think a 50/60bp-long (total) oligo (PS modified) , with your point mutations, should be enough. If you want to use classical non modified (PO) oligos, you should extent the extremities to improve the efficiency.
And try if possible to insert the restriction sites in the mutations you induce, just to make the screenign easy.
Maybe, you should also mutate the PAM, for your sgRNA not tu cut after the insertion of your mutations..
Thanks for your suggestions and for your paper, everything was extremely helpful!
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