Hello,
Does anyone know if it's possible to use this construct to clone two gRNAs targeting two genes? If yes, is there a protocol or reference?
Thanks,
Abdalla
Hi Abdalla,
There is just one BbsI cloning site for 1 gRNA sequence in this vector.
But you can clone your two gRNAs in PX459, co transfect both PX459 gRNA geneA and PX459 gRNA geneB vectors and select your cells with puromycin.
To see if you have simultaneous knockdown of your 2 genes of interest, extract gDNA of transfected and untransfected cells, amplify targeted regions by PCR and sequence PCR products with both fwd and reverse primers. When gRNA efficiently induced DSB, you shoud see a blurry chromatogram from the cut site on (see picture).
Although puromycin selection won't distinguish cells transfected with just one or the two plasmids, you can have good hopes that puromycin-resistant cells have both. (from my experience, i used to co transfect plasmids expressing GFP and mCherry respectively to double select 293T cells by FACS and cells mostly had signal for both or none)
Otherwise you'll have to first do geneA kd, select and then do the same for geneB
And beware of off-targets :)
Good luck!
Daniel
Hi Daniel,
Thanks a lot for your answer, I have been doing some search and came across this paper that developed a way to simultaneously knocking out different genes using the same vector. It's a lot more tedious but it will make my life easier. As I need to knockout various genes and overexpress others. If I use GFP and Puro crispR constructs I'll end up with many controls.
Here is the paper in case you are interested in trying it (doi:10.1007/s00018-016-2271-5).
Abdalla
Hi Abdalla,
Can I have the title of the paper, please?
Many Thanks,
Batool
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