A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

1. Culturing HEK-293T Cells and Seeding Cells for Transfection

NOTE: Human Embryonic Kidney 293T (HEK-293T) cells are grown in DMEM, high glucose media supplemented with 10% bovine calf serum supplemented with iron and growth promoters, and 1x antibiotic-antimycotic solution (100x solution contains 10,000 units penicillin, 10 mg streptomycin and 25 µg amphotericin B per mL). The media is also supplemented with 1x sodium pyruvate, 1x non-essential amino acid mix, and 2 mM L-Glutamine (stock 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl). Cells are cultured in 100 mm tissue culture plates (approximate growth surface area is 55 cm²). A sub-cultivation ratio of 1:10 is used with sub-culturing every 2 – 3 days. Trypsin-EDTA 0.05% is used for dissociation of cells between passages. To maintain consistency between experiments, we recommend testing calf sera when switching to a different lot/batch and monitor any changes in cell growth, transfection efficiency, and vector production.

1. Start a new culture by using low passage cells (it is recommended to not use cells after passage 15 or if growth slows down) by seeding into a 10-cm tissue culture plate. Use DMEM supplemented with 10% serum for cell growth. Grow cells at 37 °C with 5% CO₂ in a standard tissue-culture incubator. Use a standard hemocytometer to count cells for all subsequent steps.
2. Once the cells reach 90 – 95% confluent growth, reseed into 15 cm tissue culture plates (below, Steps 1.3 – 1.5).
3. To reseed, aspirate media from the confluent plate and gently rinse with sterile 1x PBS. Incubate cells with 2 mL of a dissociation reagent (e.g., Trypsin-EDTA) at 37 °C for 3 – 5 min. Add 8 mL of media containing serum to inactivate the dissociation reagent, and triturate 10 – 15 times with a 10 mL serological pipette to create a single cell suspension. Resuspend cells in the culture media to obtain a cell density of approximately 4 x 10⁶ cells/mL.
4. To enhance adherence to the substrate, pre-coat the 15 cm plates with 0.2% gelatin, adding 8 mL of gelatin per plate. Spread evenly across the surface of the plate, incubate at room temperature for 10 min, and siphon off the liquid.
5. Bring the total volume of each plate to 25 mL with warm (37 °C) HEK-293 T media (see Note under Step 1) and seed the plates by adding 2.5 mL of cells (total ~1 x 10⁷ cells/plate). Incubate plates at 37 °C with 5% CO₂ overnight or until 70 – 80% confluency is reached.
   NOTE: Up to six 15 cm plates can be used for production. Adjust volume of media per plate to ~20 – 22 mL when using more than four plates (refer to Step 5 of the protocol for rationale).

2. Transfecting HEK-293T Cells Using a Calcium Phosphate-based Protocol

1. Transfection reagents
   1. To prepare 2x BES-buffered solution BBS (50 mM BES, 280 mM NaCl, 1.5 mM Na₂HPO₄), combine 16.36 g of NaCl, 10.65 g of BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid), and 0.21 g of Na₂HPO₄. Add double-distilled H₂O (dd-H₂O) up to 900 mL. Dissolve, titrate to pH 6.95 with 1M NaOH, and bring volume to 1 L. Filter via 0.22 µM filter unit. Store at -20 °C.
   2. Prepare 1M CaCl₂. Filter solution via a 0.22 µM filter. Store at 4 °C.
2. Observe the plates that were seeded a day prior. Cells are ready for transfection once they reach 70 – 80% confluency.
3. Aspirate old media from the plates and gently add freshly-prepared media without serum.
   NOTE: See Supplementary File 1-Plasmids for details about the chosen plasmids and their preparation.
4. Prepare the plasmid mix by aliquoting the four plasmids into a 15-mL conical tube. For a single 15-cm dish preparation, use 37.5 µg of the CRISPR/Cas9-transfer vector (pBK198 or pBK189), 25 µg of pBK43 (psPAX2-D64E), 12.5 µg pMD2.G, and 6.25 µg of pREV (Figure 1c).
5. Add 312.5 µL 1M CaCl₂ to the plasmid mix. To this, add up to 1.25 mL of sterile dd-H₂0.
6. Slowly (drop-wise) add 1.25 mL of 2x BBS solution while vortexing the mix. Incubate for 30 min at room temperature.
7. Add the transfection mixture dropwise to each 15-cm plate (2.5 mL per plate). Swirl the plates gently and incubate at 37 °C with 5% CO₂ for 2 – 3 h. Thereafter, add 2.5 mL (10%) serum per plate and continue incubating overnight (12 – 18 h).
   NOTE: Some labs use 3% CO₂ incubators to stabilize media pH. However, we do not observe any difference in transfection efficiency between 3% and 5% CO₂. Also, the size of CaPO₄ precipitates is crucial for transfection efficiency; the transfection mix has to be clear prior to its addition onto the cells. If the mix becomes cloudy during incubation, prepare fresh 2x BBS (pH = 6.95).

Figure 2: Concentration of the viral particles using double-sucrose gradient protocol. Viral particles collected from the supernatant (SN) are loaded onto gradient sucrose gradient. 70%, 60%, 30%, and 20% sucrose solutions are used to create the gradient. Following centrifugation, the particles collected from the 30 – 60% sucrose fractions are further loaded onto a 20% sucrose cushion and precipitated. The final pellet containing purified viral particles is resuspended in 1x PBS for further usage (see text for further details). Please click here to view a larger version of this figure.

3. Day After Transfection

1. Observe the cells to ensure that they are nearing 100% confluency at this point with little to no cell death. Replace media by adding 25 mL of fresh DMEM + 10% serum to each plate. Continue incubating at 37 °C with 5% CO₂ for an additional 48 h.
   NOTE: Adjust volume of media in step 3.1 if using more than six 15 cm plates (see Step 5.2, Note).

4. Harvesting Virus

1. Carefully collect the supernatant from all the tissue culture plates containing the transfected cells using a sterile 10 mL tissue culture pipette and pool in 50 mL centrifuge tubes.
2. Clear the suspension by centrifugation at 400 – 450 x g for 10 min using a tabletop centrifuge. Filter the supernatant through a 0.45 µm vacuum filter unit.
   NOTE: The filtered supernatant can be stored at 4 °C for up to 4 days before proceeding with concentration, or aliquoted and stored at -80 °C. The expected titers of IDLV-Sp1-CRISPR/Cas9 (non-concentrated) viral preparations should be ~2 x 10⁷ TU/mL (refer to Step 6 for titer determination). However, avoid subjecting the viral preparations to multiple freeze-thaw cycles, as each round of freezing and thawing results in a 10 – 20% loss in functional titers.

5. Concentration of Viral Particles by Ultracentrifugation

NOTE: We utilize a double-sucrose method of purification that involves two steps: a sucrose gradient step and a sucrose cushion step (Figure 2).

1. Load the conical ultracentrifugation tubes in the following order to create a sucrose gradient: 0.5 mL 70% sucrose (dissolved 1x PBS), 0.5 mL 60% sucrose (dissolved in DMEM), 1 mL 30% sucrose (dissolved in DMEM), and 2 mL 20% sucrose (dissolved in 1x PBS).
2. Add the virus-containing supernatant carefully to the gradient. As the total volume of supernatant from four 15 cm plates is 100 mL, use six ultracentrifugation tubes per spin to process the full volume of the virus supernatant.
   1. Each ultracentrifugation tube used for this step has a volume capacity of up to 30 mL (including the volume of sucrose), distribute viral supernatant equally among tubes, leaving at least 10% headspace to prevent spillage.
   2. Adjust the culture volume to ~20 – 22 mL per plate when using more than four 15 cm plates for each experiment, so that the final volume of the pooled viral supernatant can easily be accommodated within six ultracentrifugation tubes.
   3. Fill ultracentrifugation tubes to at least three-fourths their total volume capacity, otherwise breakage of tubes can occur during centrifugation, resulting in possible loss of sample and/or equipment damage.
3. Balance the tubes with 1x PBS and centrifuge samples at 70,000 x g for 2 h at 17 °C (see Table of Materials for rotor details).
   NOTE: To prevent disruption of the sucrose layer during acceleration, set the ultracentrifuge to slowly accelerate the rotor to 200 rpm during the first 3 min of the spin. Similarly, set the ultracentrifuge to slowly decelerate the rotor from 200 rpm to 0 rpm over 3 min at the end of the spin.
4. Carefully collect 30 – 60% sucrose fractions into clean tubes (Figure 2). Add cold 1x PBS to the pooled fractions and bring up the volume to 100 mL; mix by pipetting up and down several times.
5. Proceed to the sucrose cushion step by carefully layering the viral preparation on a sucrose cushion. For this, add 4 mL of 20% sucrose (in 1x PBS) to the tube, followed by ~20 – 25 mL of the viral solution per tube. If tubes are less than three-fourths full, top up with sterile 1x PBS.
6. Carefully balance and centrifuge the samples at 70,000 x g for 2 h at 17 °C, as before. Pour off the supernatant and allow the remaining liquid to drain by inverting tubes on paper towels.
7. Aspirate remaining droplets in order to remove all liquid from the pellet. At this step, the virus-containing pellets should be barely visible as small translucent spots.
8. Resuspend pellets by adding 70 µL of 1x PBS to the first tube and thoroughly pipetting the suspension, subsequently transferring the suspension to the next tube and mixing as before, continuing until all the pellets are resuspended.
9. Rinse the tubes with an additional 50 µL of cold 1x PBS and mix as before. Ensure that the combined volume of the final suspension is ~120 µL, and appears slightly milky; clear it by centrifugation at 10,000 x g for 30 s on a tabletop microcentrifuge.
10. Transfer the supernatant to a fresh microfuge tube, make 10 µL aliquots, and store them at -80 °C.
    NOTE: Avoid performing repeated freeze-thaw cycles on the lentiviral samples. Except when centrifugation is required, efforts should be made to carry out the remaining steps in tissue culture hoods, or designated tissue-culture rooms using appropriate biosafety measures (see Discussion).

6. Estimation of Viral Titers

1. p²⁴ -enzyme-linked immunosorbent assay (ELISA) method
   NOTE: The assay is carried out using high-binding 96-well plates as per the instructions of the NIH AIDS Vaccine Program for HIV-1 p²⁴ Antigen Capture Assay Kit (see Table of Materials) with modifications²⁹.
   1. The next day, wash the wells three times with 200 µL 0.05% Tween 20 in cold PBS (PBS-T solution). Coat the plate with 100 µL of monoclonal anti-p²⁴ antibody at a dilution of 1:1500 in 1x PBS and incubate overnight at 4 °C.
   2. To eliminate non-specific binding, block the plate with 200 µL 1% BSA in PBS; wash three times with 200 µL 0.05% Tween 20 in cold PBS (PBS-T solution) for at least 1 h at room temperature.
   3. Prepare samples: For concentrated vector preparations, dilute 1 µL of the sample 100-fold by adding 89 µL of dd-H₂0 and 10 µL of Triton X-100 (final concentration of 10%). For non-concentrated preparations, prepare ten-fold diluted samples (add 80 µL of dd-H₂0 and 10 µL of Triton X-100 (final concentration of 10%) to 10 µL of the sample).
      NOTE: Samples can be stored at -20 °C at this step for an extended period of time for later usage.
   4. Prepare HIV-1 standards by applying a 2-fold serial dilution (with a starting concentration 5 ng/mL).
   5. Dilute concentrated samples (from 1:100 pre-diluted stocks) in RPMI 1640 supplemented with 0.2% Tween 20 and 1% BSA to establish 1:10,000, 1:50,000, and 1:250,000 dilutions. Dilute non-concentrated samples (from 1:10 pre-diluted stocks) in RPMI 1640 supplemented with 0.2% Tween 20 and 1% BSA to establish 1:500, 1:2500, and 1:12,500 dilutions.
   6. Apply samples on the plate in triplicates and incubate overnight at 4 °C.
   7. The next day, wash the wells six times and incubate at 37 °C for 4 h with 100 µL polyclonal rabbit anti-p²⁴ antibody, diluted 1:1000 in RPMI 1640, 10% FBS, 0.25% BSA, and 2% normal mouse serum (NMS).
   8. Wash six times as above and incubate at 37 °C for 1 h with goat anti-rabbit horseradish peroxidase IgG diluted 1:10,000 in RPMI 1640 supplemented with 5% normal goat serum, 2% NMS, 0.25% BSA, and 0.01% Tween 20.
   9. Wash the plate, as above, and incubate with TMB peroxidase substrate at room temperature for 15 min.
   10. Stop the reaction by adding 100 µL of 1 N HCL. Measure sample absorbance at 450 nm using the absorbance plate reader.
2. Measurement of fluorescent reporter intensity
   1. FACS Method
      NOTE: The extent of GFP signal depletion in cells can be accurately estimated by measuring mean fluorescence intensity of the transduced cells via flow cytometry. Please refer to the recent paper ²⁸ for FACS data analysis, presentation, and interpretation. The protocol is described as follows.
      1. Make a ten-fold serial dilution of the preparation (from 10^-1 to 10^-5) of the viral preparation in 1x PBS.
      2. Seed approximately 5 x 10⁵ 293T cells in each well of a 6-well plate in a final volume of 2 mL per well. Add 10 µL of each viral dilution to the cells and incubate cells at 37 °C for 48 h.
      3. Harvest cells for FACS analysis as follows: add 200 µL of 0.05% Trypsin-EDTA solution and incubate cells at 37 °C for 5 min. Add 2 mL of a complete DMEM media and collect samples into 15 mL conical tubes.
      4. Pellet cells by centrifugation at 400 x g at 4 °C, and resuspend the pellet in 500 µL of cold 1x PBS.
      5. For fixation, add an equal volume of 4% formaldehyde solution to this suspension and incubate for 10 min at room temperature.
      6. Pellet fixed cells and resuspend in 1 mL of 1x PBS. Analyze GFP expression using a FACS instrument, as described in Ortinski et al.²⁸ Briefly, use the following formula to determine the virus' functional titer:
         
         NOTE: Here Tg = number of GFP-positive cells counted; Tn = total number of cells counted; N = total number of cells transduced; V = volume (in µL) used for transduction. For example: If 1 x 10⁶ cells were transduced with 10 µL of virus, 2 x 10⁴ cells were counted and 5 x 10³ were GFP-positive, based on the above equation the functional titer would be:
         
   2. Counting GFP-positive cells
      1. Calculate the multiplicity of infection (MOI) used for transduction. Test a broad range of MOIs (1 – 10), with increasing MOIs resulting in a higher transduction efficiency.
      2. Prior to transfection, seed a 6-well plate with approximately 3 – 4 x 10⁵ cells per well. Once the cells reach >90% confluency (usually within 24 h), transduce with the purified virus at pre-determined MOIs.
      3. Incubate plate at 37 °C with 5% CO₂ in a standard tissue culture, and monitor cells at regular intervals for 1 – 7 days for changes in GFP signal.
      4. Count the number of GFP-positive cells with a fluorescent microscope (PLAN 4X objective, 0.1 N.A, 40X magnification) fitted with a GFP filter set (excitation wavelength-470 nm, emission wavelength-525 nm), using naïve (un-transduced) cells to set the population of GFP-negative and positive cells.
      5. Estimate the final titer by adjusting for the dilution factor and the volume, using the following formula:
         
         NOTE: Here, N = number of GFP-positive cells, D = dilution factor, M = magnification factor (usually 20X), V = volume of virus used for transduction. For example, for 20 GFP-positive cells (N) counted at a dilution of 10^-4 (1:10,000) in a 10 µL sample (V) at 20X magnification (M) (D) would result in a functional titer of (20 x 10⁴) x (20) x (10) x (100*) = 4 x 10⁸ TU/mL.
         (*to adjust to per mL)