I am working on some Gastric cancer cell lines, and i want to use lentiviral based crispr cas9 technology to knockout a gene from GC cell lines, previously the problem was mainly in the poor transfection of the cell lines so this time i want to use Lentiviral ( previously we used pX459 vector for CRISPR cas9. Can anybody suggests me how to design or get a vector that contains PURO gene and also can deliver dual guide RNAs, and only one vector can be used.Thanks in advance!
Ruhi Anjum pLentiCRISPR-E plasmid (#78852) for this purpose can be requested from Addgene. To get started, simply request the plasmid from Addgene and follow their instructions for cloning and gRNA selection.
all the best!
@vipin Bhardwaj
Thank you so much for your suggestion but actually my problem is that i want a vector in which i can insert two guide RNAs, hence it should contain two gRNAs scaffold ( dual guide RNA). I tried to search from addgene
In addgene i found lentiCRISPR plasmids with puro gene and 1 gRNA scaffold only. No idea how to design a single vector with two gRNA inserts+ puro
You can clone two guide RNAs separately. Generate lentiviral particles and then co-transduce them together in your target cells. That should solve the problem if you are not getting a vector with two gRNA scaffolds.
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