Hi everyone, we recently started using CRISPRi and CRISPRa in our lab and we are struggling to get good results - mainly with CRISRPi. For the gRNAs we used 5 different gRNAs alone or in combination, designed with the Broad Institute sgRNA design tool or the sgRNA library published by Gilbert et al., Cell 2014. I made a stable dCas9-KRAB Hela cell line (confirmed by WB and validated using control gRNAs) and did several time courses using transient transfection (Lipo3000) for delivering our gRNAs. However, we could not observe a significant effect of our gRNAs on the expression level of our target genes. We haven't tried the lentiviral approach yet due to limitations in the lab but my question is now if we ever obtain a good result with using transient transfections?
Looking forward to some input.
Thanks,
Verena
Just a quick question, what is your transfection efficiency in your cells, and what kind of controls did you use?
For a quick test, I would do a selection after you transfect your gRNAs and then test your knockdown (and I would wait at least 48 hours).
Hi Nicolas, we are not 100% sure about the transfection efficiency as our gRNA plasmid has no fluorescent marker. Additionally our dCas9 and gRNA plasmid have the same selection marker. We recently ordered a different gRNA plasmid with a different selection marker to try what you've suggested. H
However, we know paused this approach and try to do it using the lentiviral system.
Hi Verena have you got any updates on whether the lentivirus worked better? Did you manage to get the transient transfections to work?
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