Experiment Summary

The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and high success rate. However, recent data suggests poxvirus genomes are not repaired well following Cas9 cleavage. As a result, CRISPR/Cas9 is inefficient as an editing tool, but very effective as a programmable selection agent. Here, we describe protocols for the generation and enrichment of recombinant vaccinia viruses using targeted Cas9 as a selection tool. This novel use of Cas9 is a simple addition to current homologous recombination-based methods that are widespread in the field, facilitating implementation in laboratories already working with poxviruses. This is also the first method that allows for isolation of new vaccinia viruses in less than a fortnight, without the need to incorporate a marker gene or manipulation of large poxvirus genomes in vitro and reactivation with helper viruses. Whilst this protocol describes applications for laboratory strains of vaccinia virus, it should be readily adaptable to other poxviruses.

Fig. 1 Workflow for generation of recombinant VACVs using Cas9 selection.

Materials and Reagents

1. 6-well plates, flat bottom
2. 96-well plates, flat bottom
3. Pipette tips
4. Serological pipette
5. 2.0 ml cryogenic vials
6. 2.0 ml Sarstedt tubes
7. 5 ml Sarstedt tubes
8. 30 ml Sarstedt tubes
9. 8-strip Sarstedt PCR tubes
10. Dulbecco's Modified Eagle's Medium – high glucose
11. Dulbecco's Modified Eagle's Medium – high glucose, no glutamine, no phenol red
12. L-Glutamine (200 mM)
13. Fetal bovine serum (FBS)
14. Trypsin-EDTA (0.5%), no phenol red
15. Dulbecco's Phosphate Buffered Saline
16. LipofectamineTM 2000
17. 293A (Human embryonic kidney epithelial) cells
18. BS-C-1 (African Green monkey kidney epithelial) cells
19. VACV WR, often referred to as Western Reserve. Other replication competent strains should behave similarly.
20. VACV mCherry (derivative of VACV WR)
21. Cas9 nuclease, S. pyogenes with 10× Cas9 nuclease reaction buffer
22. gRNAs purchased as Ultramer® RNA Oligos, stored at -80°C
23. Plasmids for recombination synthesized prior to experimentation and stored at -20°C
24. Plasmid Mini Kit
25. Carboxymethyl cellulose (CMC) sodium salt
26. 100% ethanol
27. Sharpie® Ultra Fine Permanent Marker
28. Taq DNA polymerase with Buffer
29. Proteinase K (Fungal)
30. Stocks are typically made at 10 mg/ml in 50 mM Tris pH 8 and stored at -20°C and dilutions are made in nuclease-free water.
31. dNTP Mix
32. PCR primers purchased as Custom DNA oligos and stored at -20°C
33. Gel loading dye, Purple (6×)
34. UltraPure Agarose
35. SYBRTM Safe DNA Gel Stain
36. D0 media
37. D2 media
38. D2 media - no phenol red
39. D10 media
40. D10 media - no phenol red
41. 4% CMC/D2 - no phenol red

Equipment

1. Class 2 biosafety cabinet
2. Incubator with CO₂
3. -80°C freezer
4. Branson Analog Sonifier S-450 Sonicator
5. Inverted microscope
6. Dry-block heater with digital control
7. PCR thermocycler
8. Table-top centrifuge

Procedure

A. Infection and transfection

1. Perform all work in a class 2 biosafety cabinet.
2. Seed 1 × 10⁶ 293A cells/well in 2 ml D10 media in a 6-well plate.
3. Incubate cells overnight at 37°C, 5% CO₂.
4. The next day, thaw VACV stock at room temperature.
5. Sonicate virus stock 3 times (20 s each, 5 s break in between) on ice.
6. Dilute virus stock to 5 × 10⁴ pfu/ml in D0 media, allowing 1 ml for each well. This gives in the order of 0.05 pfu/cell, based on the number of cells plated.
7. Aspirate media from 293A cells and replace with diluted virus.
8. Incubate cells for 1 h at 37°C, 5% CO₂.
9. Dilute 2 µg recombinant plasmid DNA in 120 µl D0 media and incubate at room temperature for 10 min (Table 1). Label tube "Mix 1".

   Recommended: It is advised to perform transfections in duplicate, so as to have two distinct lineages of virus and ensure acquisition of desired recombinant.

   Table 1. Preparation of DNA for transfection (Mix 1)

   Component	Volume(µl)
   	Per well	3 wells
   Plasmid DNA	2 µg	6 µg
   D0 media	Up to 120	Up to 360
   Total	120	360

10. Prepare "Mix 2" by adding 4 µl Lipofectamine^TM 2000 reagent to 200 µl D0 media and leave for 5 min (Table 2).

    Table 2. Preparation of Lipofectamine^TM 2000 mixture (Mix 2)

    Component	Volume(µl)
    	Per well	3 wells
    LipofectamineTM 2000	4	12
    D0 media	196	588
    Total	200	600

11. Combine "Mix 1" and "Mix 2" to a final volume of 320 µl/well and incubate at room temperature for 20-30 min.
12. After 1 h incubation, remove virus-containing media from cells and discard.
13. Add 680 µl fresh D0 media to each well.
14. In a dropwise manner, add 320 µl of DNA/Lipofectamine^TM mix to each well and rock the plate gently.
15. Incubate plate for 4 h at 37°C, 5% CO₂.
16. Remove media and replace with 2 ml D2 media.
17. Incubate plate for 24 h at 37°C, 5% CO₂.
18. At 24 h post-infection, collect cells and supernatant by pipetting up and down vigorously and transferring to a 2 ml cryogenic vial.
19. Freeze cells and supernatant rapidly using a dry-ice/ethanol bath, and thaw using a 37°C waterbath or heat block. Cycle through freezing and thawing three times to release virus (or store at -80°C till ready for Cas9 selection).
20. Proceed to Section B for selection of recombinant viruses.

B. Cas9 selection

1. Perform all work in a class 2 biosafety cabinet.
2. Seed 1 × 10⁶ 293A cells/well in a 6-well plate and incubate overnight at 37°C, 5% CO₂.
3. Allow for 6 wells per tube of collected cells from A.
4. For selection, prepare ribonucleoprotein (RNP) complexes of Cas9 and gRNA by mixing the components in Table 3.

   Table 3. Preparation of Cas9/gRNA RNP complexes

   Component	Volume(µl)
   	Per well	6 wells
   Cas9 nuclease, S. pyogenes	0.67	4
   10× Cas9 nuclease reaction buffer	0.67	4
   gRNA (2 µg/µl)	1.00	6
   Nuclease-free water	4.33	26
   Total	6.67	40

5. Incubate Cas9/gRNA RNP mix for 10 min at room temperature, then add D0 media for a total of 120 µl per well (e.g., for 6 wells this is 680 µl of D0, for a total volume of 720 µl).
6. During the incubation in step B4, prepare Lipofectamine^TM 2000 mix as in Table 2 to a final volume of 200 µl per well.
7. Combine Cas9/gRNA mix and Lipofectamine^TM 2000 giving a final volume of 320 µl (120 µl RNP + 200 µl Lipofectamine^TM 2000) per well and let sit for 20-30 min.
8. Remove media from cells and replace with 680 µl fresh D0 media.
9. In a dropwise manner, add 320 µl of Cas9/Lipofectamine^TM 2000 transfection mix to each well.
10. Incubate for 4 h at 37°C, 5% CO₂.
11. Using the freeze-thawed tubes of virus from A (which should include the desired recombinant), sonicate 3 times for 20 s each on ice, with 5 s breaks.
12. Prepare 1/5 serial dilutions of virus in 1.25 ml D0 media, changing tip between each tube. Start with a 1/25 dilution (Figure 2).

    Fig. 2 Preparation of 1/5 serial dilutions for plating out virus.

13. Aspirate media from cells at 4 h post-transfection and add 1 ml of each virus dilution.
14. Incubate for 1 h at 37°C, 5% CO₂.
15. Replace virus with 2 ml fresh D2 media (or D2 without phenol red if you wish to observe fluorescence) and incubate for 48 h at 37°C, 5% CO₂.
16. Use an inverted microscope to look for evidence of virus infection. Starting from the most- to least-infected, identify the first well in which the CPE is not uniform across the monolayer (Figure 3). Collect cells and supernatant from this well by pipetting up and down vigorously (scraping first if necessary) and transfer to a 2 ml cryogenic vial.

    Optional: Wells on either side can be harvested similarly and kept as back-ups.

    Fig. 3 Cytopathic effect as an indicator of appropriate well selection at 48 h post-VACV infection.

17. Freeze-thaw 3 times and sonicate 3 times as above.
18. Prepare new plates of Cas9/gRNA-transfected 293A cells by repeating steps B1-B9.
19. Use virus collected in Step B16 to prepare serial 5-fold dilutions in 1.25 ml D0 media, starting with a 1/25 dilution (Figure 2).
20. At 4 h post-transfection, remove media and replace with 1 ml of each virus dilution.
21. Complete the incubations and harvesting of virus as in steps B13-B16.
22. Freeze stocks at -80°C until ready to use.
23. Proceed to Section C for identification and purification of recombinants.

C. Isolation of recombinant viruses

1. Perform all work in a class 2 biosafety cabinet.
2. Plate BS-C-1 cells to confluency in a 6-well plate in 2 ml D10 media (6 wells per plaque to be tested; see Note 5).
3. The following day, take virus collected after 2 rounds of Cas9 selection and sonicate 3 times as above.
4. Serially dilute collected virus 1/10 in 1.2 ml D0 media.
5. Remove media from cells and add 1 ml virus stock to each well.
6. Incubate plate at 37°C for 1 h, 5% CO₂.
7. After 1 h, aspirate media and replace with 2 ml 4% CMC/D2.
8. Incubate for 48 h at 37°C, 5% CO₂.
9. Locate plaques using an inverted microscope. If the desired recombinant has fluorescent or other visible markers, use an appropriate method to identify plaques that contain the marker.
10. Mark location of desired plaques using an ultra-fine permanent marker on the bottom of the 6-well plate.
11. Return plate to biosafety cabinet.
12. Insert a pipette into the well above the marked area and aspirate 10 µl, whilst scraping to collect virus from a plaque. Virus collected by this method is referred to hereafter as a "plaque isolate".
13. Transfer plaque isolate to a 2 ml Sarstedt tube containing 500 µl D10 media.
14. Repeat for all marked plaques.
15. Plaques can be stored at -80°C.
16. Proceed to Section D to screen these isolates by PCR (necessary for marker-free recombinants), or directly to Section E if a visual marker allows identification of plaques with recombinant virus.

D. Screening of plaque isolates by PCR (marker-free recombinants)

1. Plate BS-C-1 cells to confluency in wells of a 96-well plate (one well per plaque to be tested).
2. Take 10-50 µl from each plaque isolate in C and add to 200 µl D2 media.
3. Remove media from cells and replace with the diluted virus.
4. Incubate plate for 3 days at 37°C, 5% CO₂.
5. Examine cells for presence of CPE and/or expression of markers under a microscope.
6. Remove media from wells with CPE and wash with 150 µl PBS, without disturbing cells.
7. Lyse cells by adding 10 µg/ml Proteinase K in 100 µl 1×buffer to each well and incubating for 5 minutes at room temperature.
8. Freeze plate at -80°C, then thaw at room temperature.
9. Heat samples using a 56°C heating block for 20 min.
10. Heat samples at 85°C for 10 min to inactivate Proteinase K, using a heating block.
11. Take 2 µl of each sample to perform a PCR.
12. Design primers to distinguish recombinant and parent viruses (see example in Figure 4).

    Fig. 4 Confirming identity of recombinant viruses by PCR.

13. Prepare PCR mixtures as follows (Table 4):

    Table 4. Preparation of PCR master mix

    Component	Volume(µl)
    	Per reaction	4 reactions
    10×buffer	2.5	10
    dNTPs	0.5	2
    Primers (10 µM stock)	0.5 (each)	2 (each)
    Template DNA	2	-
    Taq DNA polymerase	0.125	1
    Nuclease-free water	Up to 25	Up to 25
    Total	25	100

14. Perform PCRs using a thermocycler set to conditions appropriate for amplification.
15. Run 10 µl of each sample with 2 µl 6× Loading dye on a 1-2% UltraPure^TM agarose gel with SYBRTM Safe staining to check for DNA amplification.
16. Optional: If plaques after marker-screening or PCR only show the desired virus, or if the edit to the genome is too small to be detected by a differential set of PCR primers, sequencing of PCR products that include the edited region of the genome can be done to further validate or identify plaques of interest.

E. Proceed to Section E for purification of recombinant viruses.

1. Further plaque purification to obtain recombinant virus seed stocks