Hi Enrique,

I have requested and succeed using these plasmids for doing CRISPR knockout: https://www.addgene.org/48139/

https://www.addgene.org/48138/ (same with GFP instead of puro). To avoid sorting, i will use the first one.

We transfected our cells (mESCs) with two of this ones, each one of them with a different sgRNA. To design the sgRNAs, the best place I know is http://crispr.mit.edu/ ; just give the sequence of interest and will find the best guides the program. After designing them, I just annealed them with the digested vector and sequence before the transfection.

Good luck ;)

Hi Enrique, I agree with Anchel. You can buy plasmids from Addgene, which is a non-profit website selling plasmids made by labs world-wide. Each plasmid is publication-based.

Thanks to both of you. But I was wondering if someone used commercial predesign CRISPR plasmids (i.e. Sigma), or kits.

Thanks again.

Hi Enry,

I guess Sylwia from your former lab used commercial CRISPR plasmids.

Best,

K

Thanks Konstantin and Sylwia. Sylwia is that what I wanted, if someone used already commercial without subcloning. At the end I went for sigma formats, both plasmids and using Cas9 exogenous protein.