A few tings to check:
1. is your gRNA targeting the same region of the locus as your homology arms?
2. did you interfere with some regulatory sequence when replacing the puro with GFP?
Personally I found I get better results when I put less of the gRNA vector compared to the Cas9, usually 20-50ng gRNA/ 2µg Cas9.
Integration may also be improved by linerizing the donor prior to transfection.
Since integration is going to be rare finding a positive cell in the sea of negative ones in the absence of selection may be difficult so you may want to reconsider forgoing it and include a selection (not necessarily puro) in you construct.
Thank you very much for your reply. In answer to your questions, (1) yes the gRNA definitely targets the right region. (2) I don't think I did because the exact puro sequence was lifted out and replaced with the sequence to my gene/GFP. The surrounding sequence was not altered at all.
I have been thinking that maybe the GFP signal might not be visible if there are any positive cells. The GFP is coming from the PPP1R12C promoter, there isn't a promoter for it in my construct. In this paper they used promoter-less GFP but detected success by PCR and FACS methods.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909576/
I am looking into this more. Thank you for your advice on transfection, I will give these tips a go and hopefully have some success.
Hi Laura,
My thesis project consists on performing the same experiment, and I'm also alone on it. I have to express my gene of interest in the AAVS1 locus. I’m also using the same AAVS1 donor plasmid from addgene and using the same gRNA_AAVS1-T2 sequence for targeting the genome, however, I have the gRNA guide sequence in the plasmid px458 from addgene, which already contains the Cas9.
I maintained the puro cassette and cloned my gene of interest followed by GFP after this cassette. As you, I was expecting some cells to be resistant to puro, but I did not find any... I have the same hypothesis as you. Maybe the promoter of PPP1R12C is not strong enough. Another possibility could be the transfection efficiency. Since you are transfecting 3 plasmids at a time, the chances of a cell to get the three of them are reduced. This is the reason why I chose a plasmid which allows expressing the Cas9 and the gRNA in a single vector, by this way you increase your chances for successful transfection.
Another approach could be to stabilise the Cas9 construct in your cell line and transfect only the nude gRNA (IDT or dharmacon) and the donor vector. I think this way could significantly increase the efficiency of obtaining positive clones. I would try this in the following weeks.
Thanks very much for your reply, I am looking to improve my transfection efficiency and get a single plasmid with gRNA and cas9. I have not yet had any success and also don't get any puro resistant cells when I transfect the Addgene plasmid. Can I ask, have you had any success yet?
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