I am trying to remove a gene from primary human T cells. The recent papers from Hendel and Shumann show slightly different approaches but both directly introduced purified cas9 + guide RNA. What's the best way to disrupt genes in primary T cells? Has anyone tried using lentiviral vector instead? Thanks.
Simon Bornschein
Hey Laura,
I have done it in mouse T cells, but it should also work in human T cells. I used a lentiviral plasmid which expresses the gRNA, Cas9 and puromycin from the same construct:
https://www.addgene.org/52961/
Then you can select the transduced cells with puromycin and you end up with your knockout cells. It depends a bit on the gRNA how good your knockout will be.
Hope this helps,
Simon
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