THP-1 CRISPR protocol
Using the protocol outlined below, I am unable to detect integration of our donor construct by PCR or GFP expression in THP-1 cells. Does anyone have a protocol or a reference to the use of recombinant Cas9 protein in THP-1 cells using nucleofection, or a suggestion to improve our current protocol? THP-1 cells are recalcitrant to cationic- and lipid-based methods of transfection, meaning I was unable to use a Lipofection protocol.
Protocol for recombinant Cas9 NLS Nuclease protein to edit the genome of a THP-1 cell line:
1. Add 1 ug of Cas9 nuclease preloaded with 250 ug of validated (in HEK293M) sgRNA to form the ribonucleoprotein complex.
2. THP-1 cells are electroporated with 100 ng of endotoxin-free linearized donor DNA (a homology-directed repair template) using an Amaxa Nucleofector IIb with protocol U-001.
3. The cells were cultured for 96 hours in 1 uM SCR7, a NHEJ inhibitor.
I know THP-1 cells and monocytes in general are deficient in HDR, so any help to optimize this is appreciated!
Why do you say THP1 are deficient in HDR ("in general")?