細胞分裂に CRISPR/Cas9 を介した遺伝子ノックアウトのインテグラーゼ欠損レンチウイルスベクターの生産のためのプロトコル

The overall goal of this protocol is to describe the production and potential applications of optimized integrase-deficient lentiviral vectors capable of delivering CRISPR/Cas9 transgenes to cells in vivo and in vitro for rapid and efficient gene editing. CRISPR/Cas was a highly-efficient lab for gene editing manipulation. Here is a protocol outline in the production of a highly-concentrated optimized integrase-deficient lentiviral vectors for efficient delivery of CRISPR/Cas gene editing tools.

The technique has proven to be very efficient in the causal cell and post-mitotic neurons. Seed the HEK-293T cells a day before the transfection so that the confluency reaches between 70 to 80%the next day. Before transfection, aspirate the old media from the culture plates and add freshly-prepared media lacking serum.

In a 15-milliliter conical tube, add all the four plasmids to prepare a plasmid mix for transfecting a 15-centimeter dish. Then, add 312.5 microliters of one molar calcium chloride to the plasmid mix and adjust the volume to 1.25 milliliters with sterile double-distilled water. Keep adding drops of 2X BES buffered solution gradually while vortexing.

And then, incubate the mixture at room temperature for 30 minutes. Add 2.5 milliliters of plasmid mixture in drops to each of the 15-centimeter plates. Then, gently swirl the plates and incubate at 37 degrees Celsius with 5%carbon dioxide for two to three hours.

After three hours, add 2.5 milliliters of 10%serum to each plate and continue for overnight incubation. Use a 10-milliliter tissue culture pipette to carefully aspirate the supernatant from the transfected cells seeded on each culture dish. Then, pool the entire supernatant in 50-milliliter centrifuge tubes.

Place the tube inside a tabletop centrifuge and start the centrifugation. Then, filter the supernatant through a 0.45-micrometer vacuum filter unit. Load the conical ultracentrifugation tubes with different sucrose concentrations to form layers with sucrose gradient.

Carefully add the virus containing supernatant to the sucrose gradient. To process the entire 100-milliliter volume of viral supernatant, use six ultracentrifugation tubes in each spin. Then, balance the ultracentrifuge tubes with 1X PBS buffer and spin the samples at 70, 000 G for two hours at 17 degrees Celsius.

In clean tubes, collect the 30%and 60%sucrose fractions and add cold 1X PBS buffer to adjust the volume to 100 milliliters. Mix the samples by pipetting it up and down several times. Then, add 20 to 25 milliliters of the viral solution on top of four milliliters of 20%sucrose in 1X PBS solution to prepare the sucrose cushion.

Adjust the volume with sterile 1X PBS buffer if the tubes are less than 3/4 full and balance it. Then, spin the samples for two hours at 17 degrees Celsius. Then, pour the supernatant off and drain the remaining liquid by inverting the tubes on paper towels.

After aspirating the last remaining droplets, a pellet containing the virus is barely visible as a translucent spot. Next, resuspend the pellet in 70 microliters of 1X PBS by thoroughly pipetting the suspension. Again, transfer the suspension to another tube and thoroughly mix to completely dissolve the pellet.

Then, rinse the tube with an additional 50 microliters of cold 1X PBS buffer and mix. Keep the samples on a tabletop microcentrifuge and centrifuge at 10, 000 G for 30 seconds. After centrifugation, add 10 microliter aliquots of the supernatant to each fresh microfuge tube and store them at negative 80 degrees Celsius.

Coat a high-binding 96-well plate with 100 microliters of monoclonal anti-p24 antibody at a dilution of one to 1, 500 according to NIH AIDS vaccine program for HIV-1 p24 antigen capture assay kit. Incubate the plate overnight at four degrees Celsius. The next day, wash the plate with 200 microliters of 0.05%TWEEN-20 in cold PBS three times.

To avoid non-specific binding, block the plate with 200 microliters of 1%bovine serum albumin for one hour at room temperature. Then, wash the plate with 200 microliters of 0.05%TWEEN-20 in cold PBS three times. Then, prepare the concentrated and non-concentrated vector samples to a final concentration of 10%by diluting the vector with double-distilled water and Triton X-100.

Next, prepare the HIV-1 standards using two-fold serial dilution starting at five nanograms per milliliter. After diluting the concentrated and non-concentrated vector samples, pipette it on the plate in triplicates and incubate at four degrees Celsius overnight. The next day, wash the plate, then add 100 microliters of polyclonal rabbit anti-p24 antibody on each plate and incubate at 37 degrees Celsius for four hours.

After six washes, incubate the plate with goat anti-rabbit horseradish peroxidase at 37 degrees Celsius for one hour. After multiple washing, incubate the plate with TMB peroxidase substrate at room temperature for 15 minutes. Add one normal hydrochloric acid to stop the reaction.

And read the absorbance at 450 nanometers with an absorbance plate reader. Seed the cells in a six-well plate. Next day, when the cells reach 90%confluency, transduce with purified virus at a predetermined multiplicity of infection ranging from one to 10.

Then, incubate the plate at 37 degrees Celsius with 5%carbon dioxide. Monitor the plates to record difference in the GFP signals at regular intervals for one to seven days. Look for GFP positive cells under fluorescent microscope fitted with GFP filter set with excitation and emission wavelengths at 470 and 525 nanometers respectively.

To differentiate between the population of GFP positive and negative cells, use untransduced cells. A representative agarose gel showing the positive mutated integrase clone. The double-digested clone with restriction enzymes EcoRV and SPH1 in red-dashed box represents the mutated integrase clone.

The clone was further sequenced to check the mutation of aspartic acid at position 64 to glutamic acid. A representative bar graph for p24 ELISA assay has been shown depicting the viral titers for SP1-integrase-deficient lentiviral vector CRISPR/Cas9 plotted in the black bar and SP1-integration-competent lentiviral vectors CRISPR/Cas9 plotted in the white bar. The bar graph shows integrase-deficient and competent viral titers in the range of one times 10 to the 10.

The bar plots represent the copy number of viral particles per milliliter. Representative fluorescent images showing the mean fluorescent intensity of GFP positive cells under viral treatment. The cells were transduced with integration-competent lentiviral vectors CRISPR or integrase-deficient lentiviral vector CRISPR viral components.

A sharp reduction in GFP signal was observed at multiplicity of infection one and five respectively after transducing the integrase-competent or deficient lentiviral components with respect to the untransduced control cells. The main advantage of this technique stem from the ability of integrase-deficient lentiviral vectors to a deliver genome editing tool transiently which significantly enhance its specificity and the safety. If properly conducted, the protocols should take one week to complete.

Conducting this protocol, one should expect to generate highly-concentrated lentiviral vectors in the range of 10 to the nine, 10 to the 10 viral genome parameter.