CRISPR Lentiviral Screening: Antibiotic Selection

Antibiotic concentrations for CRISPR library selection

Antibiotic Kill Curve

A kill curve is a dose-response experiment where cells are subjected to a range of antibiotic concentrations to determine the minimum concentration of antibiotic needed to kill all cells over a desired period of time. This step is suggested for each new cell line tested as well as when a new selection antibiotic is used. High concentrations of antibiotic are undesirable as they can lead to toxicity, even in successfully transduced populations.

Required Reagents:

• Target cells in culture
  
• Antibiotic
  • Puromycin (10 mg/mL solution), (Sigma-Aldrich, Product No. P9620)
  • G418, for neomycin resistance in mammalian cells (50mg/mL solution), (Sigma-Aldrich, Product No. G8168)
  • Blasticidin (lyophilized), (Sigma-Aldrich, Product No. 15205)
  • Hygromycin B (lyophilized), (Sigma-Aldrich, Product No. H7772)
• Complete growth media specific for the target cells

Protocol:

1. Plate cells according to normal passaging protocols for the cell line in use.
   
2. The next day, replace the media with media containing serial dilutions of the antibiotic to be used during selection:
   • G418: 0.1-2.0 mg/mL
   • Puromycin: 0.5-10 mg/mL
   • Blasticidin: 2.5-20 µg/mL
   • Hygromycin 100-500 µg/mL
3. Examine the cells daily for visual signs of toxicity.
   
4. Replace the media containing antibiotic every 2 - 3 days. Culture for 3 – 15 days depending on growth rate of the cell type and length of time that cells would typically be under selection during a normal experimental protocol.

The minimum concentration of antibiotic that causes complete cell death after the length of time that cells would typically be under selection should be used for that cell type and experiment. Some cells require passage or expansion into a larger vessel in order to avoid senescence or differentiation and continue antibiotic selection.

Multiplicity of Infection (MOI)

MOI is the number of transducing lentiviral particles that should be added to each cell in order to ensure successful integration into the cell. It is highly recommended that for each new cell type to be transduced with a specific vector, a range of MOI be tested.

Required Reagents:

• Target cells in culture
• Lentivirus particles (control or clone) in the same lentiviral vector you will be using in your experiment
• Complete growth media specific for the target cells
• Complete growth media specific for the target cells with appropriate type and concentration of antibiotic identified for your cell line and the resistance gene on the lentiviral particles you are using

Protocol:

1. Plate 1.6 x 10⁴ cells into wells of a 96-well plate with 120 µL fresh media and culture overnight.
   
2. Add control virus to cells in a range of MOIs and culture overnight
   • For most cell types, a range of 0.1 - 10 MOI is suitable.
     
   • For hard to transfect cell lines you may need to increase your range to MOI of 50 or 100.
     
3. If using antibiotic selection: apply selection media for timeline established during kill curve set up.
   
   • After selection is complete, identify well with viable cells at the lowest tested MOI value. This is the MOI to use in your future transduction experiments for this cell line and vector.
4. If using fluorescence: allow cells to culture until sufficient levels of fluorophore expression are detected.
   Identify the well with desired quantifiable fluorophore expression at the lowest tested MOI value.
   
5. To calculate the amount of virus needed after establishing MOI:
   (total number of cells per vessel or well) x (desired MOI) = total TU needed
   (total TU needed) / (TU/mL functional titer) = total mL of lentiviral particles to add to each vessel or well