Dear all! we are trying to knockdown a gene in lymphoblastoid cell line using Origene CRISPR/CAS9 kit with the HMR method using the recommended turbofectin reagent. However, we are not getting any strong positive signal under the flourscent microscope despite following the standard protocol. Some faint green signals are observed but they eventually disappear. Anyone has experience regarding this situation?
If you are using an established cell line (i.e. not primary cells) then you may consider using electroporation instead (see https://altogen.com/product/ramos-electroporation-kit-lymphoma-cells-crl1596/). I'm assuming your cell line is in a suspension, and electroporation will be better suited for transfecting the cells. Additionally, CRISPR/Cas9 has a low efficiency - 10 to 15% at max, so it's no surprise that you aren't getting a high signal. Adding nuclear localization sequences and optimizing your transfection conditions might help.
Dear Micheal Davis, thanks for the reply. Yes I am using suspension culture. I tried electroporation but did not get satisfactory results. The link you mentioned is specifically for siRNA. In cases of siRNA, the electroporation has good efficiency as it may be transient but I am facing problems in stable knockout. Additionally, regarding NLS there are contradictory views but many researchers say NLS has no substantial effect on the CAS function and localization. I am trying to optimize the strategy however, I am interested if someone has specifically performed a knockout in lymphoblastoid cell lines as the efficiency is dependent on cell line as well.
Hi Abdul,
It's been a few months since you posted the initial question, however I wanted to follow up about your CRISPR results. As mentioned before, it is very difficult to transfect LCLs, so many researchers use electroporation, or nucleofection (see Chapter CRISPR/Cas9-Mediated Genome Editing in Epstein-Barr Virus-Tr...
). However, OriGene does offer CRISPR vectors in the form of lentivirus, if you would prefer the infection route.- Badges
- Science method