Efficient Gene Editing Through an Intronic Selection Marker in Cells

Determine percentage of DsRed-positive cells from dsDNA- or ssDNA-transfected HEK293T cells determined by FACS.
Conclusion:
Our data demonstrated that a single-stranded CMV-DsRed donor led to significantly lower fluorescence intensity than a double-stranded donor HEK293T cells were obtained from the American Type Culture Collection. Cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum at 37°C and 5% CO2. Fifteen micrograms of LentiCRISPR-V2 and 2 µg of donor DNA were cotransfected into 5 X 107 cells with Lipofectamine 2000TM (Invitrogen) according to the manufacturer’s instructions. Subsequently, transfected cells were treated with 1 µg/mL puromycin for 72 hours. Cells were washed with PBS and treated with 0.05% trypsin. The cell suspension was filtered through a 40 µm cell strainer (BD Falcon) before FACS. Flow cytometry analysis and FACS were performed using BD LSR II. Cells isolated by FACS were then cultured for one week and processed by FACS again to enhance the positive rate.