Lipofectamine crisprmax cas9 plus transfection reagent

CRISPR experiments were performed as previously described⁴⁸ using Gene Knockout Kits v2 (Synthego) for human PIM1, -2, -3, and S100A8 (with each Kit containing a mixture of three target-specific, synthetic multi-guide sgRNA sequences and purified Cas9 nuclease) and Lipofectamine CRISPRMAX Cas9 Plus transfection reagent (Thermo Fisher, CMAX00008) according to the protocol (entitled “CRISPR editing of immortalized cell lines with RNPs using lipofection”) provided by Synthego. Non-targeting sgRNAs (Negative control scrambling sgRNA #1 and #2) were also purchased from Synthego and mixed at 1:1 before use. sgRNA/Cas9 reverse-transfection was performed in 24-well plates (3.9 pmol sgRNA + 3 pmol Cas9 per well), and the resulting heterogeneous populations of cells were expanded in 6-well plates. The sgRNA/Cas9-treated cells were used in downstream assays within ~six weeks of transfection to avoid the expansion of undesired cell populations. Each biological replicate was initiated by sgRNA/Cas9 transfection. We found that while MDA-MB-468 cells were readily amenable to CRISPR-based genetic manipulation, BT-20 cells and freshly isolated mouse CD11b + Gr1+ myeloid cells were not, due to transfection-induced excessive toxicities.