Hello everyone; I am new to CRISPR.
Owning to our strategy to screen positive cells, we yearn to involve two selection markers (GFP and puromycin) in the LentiCRISPRv2 vector. However, we figure out that the viral titer is low in this way, and the possible explanation is the large size of the vector. Also, we noticed that there are several non-functional sequences (?) in this vector. The main question is: Is it ok to remove the highlighted sequence (including EM7 promoter, BleoR, SV40polyA signal, lac promoter, and CAP binding site) in the file?
We think this strategy might be practical since the selection strategy in prokaryotic system in based on Ampicillin, and Zeocin (BleoR) should be the a trademarker, which is actually non-functional?
Thanks a lot
That sequence is outside LTRs. Removing it won't improve viral titer and it won't increase the lentivirus cargo size. In my opinion there's no trchnical advantage in temoving it.
Francesco Aulicino thanks for your reply. I realize that the seq I want to delete is outside LTRs. However, although it will not affect lentivirus cargo size, it may improve the Transfection efficiency during virus preparation. In this way, it might be able to improve virus titer?
Also, I want to known whether the highlighted seq is nonfunctional in this plasmid. Thanks a lot.
Lo Tzu Yu the sequence length is unlikely to influence transfection efficiency. You are planning to remove a sequence of approx 500 bp out of a 14.5 kb plasmid. This will have a minimal impact on transfection efficiency. Regarding increasing viral titer, the pLentiCRISPR has been widely used by many labs now... and if there are problems related to transfection or titer, they would have been spotted by now. So I think in terms of vector optimisation there's little to do to solve your problem.
You mention selecting transduced cells with GFP and Puro. Have you inserted a GFP expression cassette? That would definitely interfere with the packaging cargo capacity of lentivirus (8.5 to 10 kb) and bring the viral titer down.
Regarding the function of the sequence you propose to remove. I don't think it's involved in lentivirus production but it might have been used to preserve vector integrity during bacterial propagation by applying a double antibiotic selection. I am not 100% sure of that but you can contat the authors of the paper, I am sure they'll be able to provide a meaningful explanation.
Hope it helps
Francesco
Lo Tzu Yu This is the most likely cause of the reduced viral titer. Especially if the combined lenght of Ca9 GFP Puro and sgRNA cassettes exceeds the cargo capacity of the vector. To measure this, confirm that the DNA region between 5' and 3' LTR is below 8.5 kb. Bigger size will cause a reduction in viral titer and in my experience I haven't been able to package anything with a lentivector above 10 kb.
If you want to restore the size and keep eGFP and Puro, you might have to use a shorter promoter or a different (smaller) Cas variant, like CasX for instance. The latter will require also changing the sgRNA expression cassette (scaffold and spacer).
Hope it helps
Francesco
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