Production of LentiCRISPR Viruses

This protocol is modified from the Addgene website protocol

Materials

  • 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

Preparation of Cells

  • 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.

Transfection Procedure

  • 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:
Reagent pmoles ug Volume Added
psPAX2 1.3 9.2
pMD2.G 0.72 2.77
LentiCRISPRv2-sgRNA 1.64 16.1
OptiMEM - - 500 uL
  • 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.

Collecting Viral Particles

  • 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.