I'm trying to knock out a gene in mESCs. I have my guides already cloned each into a CRISPR/CAS9 plasmid also carrying GFP.
My guides didn't work after transfecting them individually, so I wish to transfect two guides, which are on the same exon, simultaneously.
In most of the method papers I find, the transfection is made either with one vector carrying the CAS9 and the guides each with its own promotor, or the CAS9 and the guides on different vectors each. My question is what is the best way to transfect two guides, and can I use my already cloned guides, with maybe an mCHERRY expression on one of the vectors? (they will then be sorted by Flow Cytometry)
Thank you for your answers!!
I assume both ways would work but I have been using RNP complexes: cas9 protein with annealed tracrRNA and guide RNA. How do yo know your individual guide didn't work? Did you make sure there is no SNPs for that targeting site? if individual guide doesn't edit (make indel), two guides might not work either. I would make sure that individual guide does cut the target and then use two guides to delete an exon.
Hope it helps!
Dear Ayellet,
I've co-transfected two sgRNAs in mESCs, each one cloned in its own px459 vector (Puro instead of eGFP) to excise whole genes. I had a similar issue since single sgRNAs were either inefficient or not able to induce KO, so I've finally opted for 2-sgRNAs approach but still using the original vectors I've made in first place.
The article is here if you need more info:
https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(20)30296-4
Hope it helps
Francesco
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