Production of LentiCRISPR Viruses

This protocol is modified from the Addgene website protocol

• Use the 293T/17 cells (purchased as a stock from ATCC and aliquoted in separate box in the liquid nitrogen; ATCC page).
• Plasmids (lenti-vector, psPAX2, pMD2.G)
• OptiMEM
• Chloroquine stocks (25 mM stocks). See Preparation of Chloroquine Stocks.
• PEI (1 mg/mL stocks). See Preparation of PEI Stocks.
• DMEM/10% FBS/PSG and DMEM/10% FBS with no PSG

• Thaw and passage cells. Use passage <20. These amounts are for one plasmid.
• Split cells normally each time seeding 7 x 10⁵ cells in a 10 cm dish, in DMEM/PSG/10% FBS.
• The day before the transfection, seed 3.8 x 10⁶ cells into a fresh plate. Seed cells into media without PSG.

• The morning of the transfection day, replace the media with fresh DMEM without PSG and containing 10 uL of 25 mM chloroquine. Wait ~5h before going onto the next step.
• Prepare DNA according to this table in a sterile 2 mL tube:

• For this amount of DNA (28 ug) try using 3:1 PEI:DNA, so add 84 uL PEI into 1 mL OptiMEM.
• Gently add the PEI solution dropwise into the DNA solution.
• Incubate at room temperature for 15-20min.
• Add transfection mixture slowly to the cells.
• Incubate overnight. The next day carefully replace with media containing PSG.

• Collect media at 96h, or at 48, 72 and 96h post infection. Combine supernatants in a 50 mL conical tube.
• Centrifuge at 500g for 5 minutes to pellet any cells.
• Filter supernatant through the 0.45 um. Aliquot and snap freeze in liquid nitrogen in screw capped vials.