The overall goal of this experiment is to improve the efficiency of the generation of mutant vaccinia virus by using CRISPR-Cas9 technology. This method can help to create new vaccinia virus vectors for biological research and medical research, in particular for gene therapy and vaccination. Viral demonstration with this method is critical, because identifying and picking up positive plaques is difficult to perform.
The main advantage of this technique is that it dramatically improves the efficiency in making our mutant the vaccinia virus. Beginning with harvested CV-1 cells, adjust their concentration to 100, 000 cells per milliliter. Then seed two milliliters of the suspension into each well of a six-well plate for transfection.
The following day, transfect the cells with 0.5 nanograms of gRNA-N2 plasmid and 0.5 nanograms of pSTCas9 plasmid. Then incubate them for 24 hours. 24 hours later, replace the medium.
Next, prepare the VVL15 virus. Dilute the backbone VVL15 virus with DMEM to 200, 000 PFU per milliliter. Then add 100 microliters of the diluted virus into each well of the transfected CV-1 cells.
Two hours later, transfect the cells with one microgram of the shuttle donor vector for expression of HR, and incubate the cells for 24 hours. The following day, detach the transfected CV-1 cells using a cell scraper. Then collect the cell suspension into a cryovial and store at negative 80 degrees celsius for future use.
To begin the first round of purifying the modified VV, seed 15 six-well plates with 300, 000 unmodified CV-1 cells per well. The next day, thaw the frozen suspension of transfected CV-1 cells at 37 degrees celsius for three minutes, and then vigorously vortex the suspension for 30 seconds to lyse the cells. Then dilute one microliter of the lysate with three milliliters of DMEM.
Add half a milliliter of this dilution to each plate well of unmodified CV-1 cells. Then return the plates to the incubator for two days. Two days later, identify RFP-positive plaques using a fluorescence microscope.
They can be spotted with a 10X objective. As they are identified, accurately label the plaques underneath the plate using a marker pen. Now prepare a cryovial containing 200 microliters of serum-free DMEM for each positive plaque to be harvested.
Next, it is important to aspirate all of the medium from the wells with labeled plaques. Then, using a 200 microliter pipette set to aspirate 30 microliters, partially load the pipette with 10 microliters of medium from the cryovial, and then scrape the plaque and draw up the cells with the remaining aspiration. Eject this into the cryovial and repeat this procedure twice more, targeting the same RFP-positive plaque to collect as many cells as possible.
Store the collections at negative 80 degrees celsius or, if the second round of purification is to be performed immediately, freeze the cryovials on dry ice for 10 minutes. The second round of purifying the modified VV is performed just like the first using the frozen cells from the first round. However, more than six RFP-positive plaques can be selected for each RFP-positive line.
Repeat the selection process until all the plaques formed from one positive plaque are RFP-positive indicating that the plaque is pure, which can take an additional three to five rounds. One day in advance, prepare a six-wall plate with half a million CV-1 cells per well. The following day, thaw the frozen purified plaque at 37 degrees celsius for three minutes.
Then vortex the plaque vigorously for 30 seconds to make a lysate. Add all of the lysate, including the cell debris, into a well of CV-1 cells, and incubate the plate for three days. Do not use the other five wells.
Inspect the cells daily under a microscope. When more than half of the cells appear cytopathic, harvest the infected cells. Ideally all the cells should be cytopathic.
Use a cell scraper to collect the cells in a 15-milliliter tube with with culture medium. Then pellet the cells at 300g for three minutes. Discard the supernatant and proceed with using PCR to verify the modification of the mutant VV, or freeze the cell pellet at negative 80 degrees celsius to do so later.
The method described improves the recombination efficiency by more than 100 fold compared to conventional protocols. For example, the VV N1LG was targeted for modification. As described, plasmas expressing Cas9, and an N1L-specific gRNA were co-transfected into the CV-1 cells to enhance their efficiency for homologous recombination.
One day post-transfection, RFP was expressed in the CV-1 cells. Next, CV-1 cells were infected with the whole lysate from the homologous recombination. RFP-positive and negative plaques were both observed in the CV-1 cells.
Following the purification protocol, a pure recombinant was obtained. All the plaques after infection with the cell lysate derived from a pure plaque with the mutant vaccinia virus presented an RFP signal. PCR was used to verify the deletion of the targeted gene.
The modified virus showed a positive signal for RFP, and no signal for N1L, unlike the intact N1L control. All were positive for the non-targeted gene A52R. After watching this video, you should have a good understanding of how to quickly make a new vaccinia virus vector.
Once mastered, this technique can be done in 10 days if it is performed properly. After its development, this technique paved the way for researchers in the field of biomedical sciences to explore new viral vectors in development of biological agents for treatment and the prevention of cancer and infectious diseases.
