Recently I used 2 CRISPR containing lentiviral for downregulating the same gene at two different exons in NSCs/NPCs. The western blot (densitometric ananlysis) showed 96% down-regulation in CRISPR1 & 84% for CRISPR2 for my gene of interest. However when I challenged those cells with Proinflammatory cytokines (in time dependent study), I found still there were some up-regulation in both CRISPR treated NSCs (result from both FACS & Western showed the same pattern).
Please kindly suggest why up-regulation of my gene of interest is still there, however before down studies, cells were in Puromycin for 12 days at 2ug/ml (although the kill curve was observed at 05.ug/ml).
Kindly suggest what could be the problem, or is it normal to still have some cells which even after CRISPR treatment can show some level of expression of the gene for which CRISPR were made.
Please kindly suggest what can I do next to get 100% knockout?
Looking forward,
Many thanks
Syed
Even in the lentiviral CRISPR systems, there can be incomplete editing, or cases where repair of the deletion does not result in disruption of the coding sequence. In addition, in some cases, mRNA for some genes can be long-lived, providing a potential reservoir for normal gene expression if your cytokine treatment kicks in at the level of translation.
If you want to increase editing efficiency, try using lenti-CRISPRs with orthogonal antibiotic selection cassettes so that you can introduce 2 gRNAs into the same cell. 84-96% editing sounds pretty good, and should be adequate to test your hypotheses, but if you combine two different gRNAs, you can probably get closer to 100%. The down-side is the higher chance of off-target effects.
Dearr Daniel,
Many thanks for your kind suggestion, although I don't have 2 different gRNAs but I used two single guided RNAs for 2 sites (exon 2 & exon 3) for the same gene and exon 2 CRISPR as intimated earlier showed a 96% KO, while exon 3 CRISPR- 84%. How about if I transfect lenti-CRISPR for exon 3 to my NPCS which are already transfected with CRISPR for exon 2, and vice versa. Can I achieve almost 100% by this way? Please kindly suggest.
Looking forward,
Many thanks,
Syed
Yes, that dual infection is what I'm suggesting, but you'll need to have two different antibiotic selection cassettes.
Without using different selection markers, there is no way to tell if a cell was infected with one or both lentis. You only want cells that are dually infected.
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