Dear Researchers!
I am facing a serious issue regarding making stable HEK293T cell line using CRISPR/Cas9. I am using paired nickase Cas9 along with cut and uncut Donor plasmid (separately). After successful antibiotic selection (for 2-weeks), I could see the signal marker (GFP) which gave me hope that the cells I got are stable and expressing my gene of interest. After doing 3' and 5' junction PCR, I got very light band (unexpectedly). I was just curious that if PCR band is so light then how Ab selection and GFP signal is so promising. Just to solve this mystery, I isolated total DNA from transfected cells and did PCR using primers specific to the backbone sequence of the Donor plasmid. Surprisingly, there was significant amplification which indicates that most of the plasmid is in non-integrated form. My concerns are:
1) Is there anyway to maximize the transgene integration using CRISPR? Should I increase the amount of nCas9 and gRNA instead of Donor?
2) The above mentioned fact will never make me able to get a stable clone having integrated transgene which is quite disappointing after a lot of effort. Is there anyway to get stable clone using HEK293T?
3) HEK293T is designed for best transfection outcome so it might not be able to degrade non-integrated plasmid. Is there anyother cell line which I can use to get stable clone. Of course that cell line must degrade the left-over non-integrated plasmid to avoid false-positive selection. I am not limited to just HEK293T so please recommend.
I will be very thankful for your kind suggestions.
Best Regards,
Nasir Javaid
Dear Nasir,
Stable plasmid integration happens at very low rate even in absence of Cas9. In fact this is a way to get stable cell lines and it was commonly used before (and I think it still is) to generate random transgene integration. In cell lines such as 293t, where transfection efficiency is higher, it is just more relevant.
Changing the cell line will not get rid of this. You will just get lower transfection efficiency but overall the rate of random plasmid integration will be similar.
In your experimental setting the sgRNAs you have picked may be just not very efficient. You can try the following:
1) Use wild-type Cas9 instead of the nickase, this will increase the rate of specific integration.
2) Make a control transfection with only your Donor, if you get the same number of eGFP expressing cells as in presence of Cas9 and sgRNAs then it means you are only getting random integrations.
3) Do not apply selection at early stages as this will increase the rate of random plasmid integration. Wait 1-2 weeks after transfection and allow the plasmid backbone to be completely diluted. At this point you can apply selection and/or sort the cells for eGFP and you should increase the chance that clones surviving your selection aquired an integration of your plasmid at early stages.
PS. Just a note, if you are usign Neomycin to select the cells I think 293t are already resistant so you will have to rely only on sorting.
Hope it helps
Francesco
Dear Francesco,
Thank you for your valuable suggestions. I am afraid using wtCas9 due to off-targets. I did control transfection and GFP expression was almost same in both the cases. Third suggestion is new and makes sense to me so I will follow that in my next experiment.
Dear Han,
Thank you for your recommendation.
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