My plan is to perform the CRISPR onto iPS cells. I want to have APP knockdown iPS cell line and differentiate them into the neuron. I do not know whereto start. I plan to order ready vector for transfection but how should I chose it? Can anybody help me?
We perform crisper in mouse embryo, cells to knockdown several different genes and what we use is a plasmid that has CAS9 and sgRNA expression in a single vector followed by an in frame GFP expression cassette. You simply need to clone your sgRNA (we have tried cloning in 2 sgRNA for one gene: increased efficiency, however even one works fine for us).
Since you are performing this in special cells (we have never tried iPSCs) you might want to have a plasmid with a selectable marker like puromycin instead of GFP. This will be best decided based on whether your cells are not sensitive to facs sorting (in which case you'll sort out positive cells by GFP) or if they are sensitive to puromycin as a selectable marker. Cas9 plasmids having either of the markers are available from the companies like adgene and all (No Conflict of interest).
Also some of our collaborators say that you get the best knockdown efficiency if your marker and cas9 expression is bicistronic. My best guess is that this is due to high expression of your GFP followed by comparatively lower expression of the second gene, in your case cas9. Resultantly, not all cells that are positive for your marker will show knock down. hence bicistronic can be a better choice (not verified by us though!)
Regarding sgRNA I would really suggest cloning in your own based on the highest scores you obtain rather than looking for a ready vector as previosuly suggested. The cloning in of sgRNA in these vectors is super easy.
You would also need to test the KD efficiency using a surveyor assay (order a commercila kit for that). I hope this gives you an overall idea. :)
pSpCas9(BB)-2A-GFP (PX458)
pSpCas9(BB)-2A-Puro (PX459) V2.0
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