Hi, we've been unsuccessful in creating single clones with deletion of a couple of proteins, after testing several sgRNA sequences for each. We are using a lentiviral vector encoding puromycin for selection marker and CAS9. What is everybody's favorite plasmid for CRISPR deletion in 3LL (LLC1) and subsequently testing the effects of the deletion in vivo?
Ideally, one would not want a lot of immunogenicity driven by the plasmid itself so that implantation/growth is not altered, although these cells form tumors almost no matter what.
Thanks!
I think that plasmid is used to amplify the number of copies of your target sequence, unless you want to over express this sequence in vivo. In any case you’ve to linearised your sequence by cutting the plasmid and then delivering it by liposome in your in vivo cell system
Unsuccessful in vitro? If so, are these proteins potentially required for cell survival/proliferation? And, could you delete other proteins with the same system? Irrespective, most of the systems I am used to only express Cas9/selection markers on non replicative plasmids, meaning that you'll loose them quickly over time in both KO and non KO clones (which you should select early based on markers + deletion, then propagate)
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