Methods

MAVERICC: Efficient marker-free rescue of vaccinia virus recombinants by in vitro CRISPR-Cas9 engineering

Cells and viruses

BSC-40 cells were obtained from American Type Culture Collection (ATCC, CRL-2761) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 11965-092) supplemented with 10% (vol/vol) fetal bovine serum (FBS, Atlanta biologicals S11150H), 1% (vol/vol) glutamine (Gibco 35050-061), and 1% (vol/vol) penicillin-streptomycin (Gibco 15140-122). The initial VACV, vNotI/tk, used in this study was a generous gift from Bernard Moss (NIH) 27. This virus is a modified version of the Western Reserve strain that contains a unique NotI restriction site in the thymidine kinase (tk) gene. The vNotI/tk virus was propagated in BSC-40 cells and harvested by collection of the cell pellet after 2–3 days of growth and three freeze/thaw cycles to release the intracellular mature virions. It was then modified to contain either mCherry or eGFP reporters under control of the p11 VACV promoter in the tk locus. rVACV-mCherry and rVACV-eGFP were generated by transfecting a custom-synthesized plasmid containing 500 bases of left- and right-homology arms flanking the mCherry or eGFP reporter, followed by three rounds of plaque purification with selection for mCherry or eGFP expression.

Fowlpox virus (FWPV) was obtained from ATCC (VR-250) and propagated in chicken embryonic fibroblasts (CEFs) also obtained from ATCC (CRL-1590). CEFs were cultured in DMEM supplemented with 2.5% (vol/vol) FBS (Atlanta biologicals S11150H), 1% (vol/vol) glutamine (Gibco 35050-061), 1% (vol/vol) penicillin-streptomycin (Gibco 15140-122) and 1 mM HEPES (Gibco). CEFs were infected with FWPV when they were about 70% confluent and virus propagation was allowed to continue for 3 days or until cytopathic effects were evident throughout the cell culture. This FWPV strain initially did not grow well in these CEFs and therefore first had to be adapted to these cells through 9 serial passages. For each passage, a 15-cm plate of CEFs was grown to 70% confluence and 10% of the viral stock from the previous passage was added to the cells. The virus was grown for 3 days before cells were harvested by scraping and subjected to 3 freeze/thaw cycles to free intracellular virions.

VACV DNA purification

VACV DNA was purified according to published methods 20,21. First, 20 15-cm plates of BSC-40 cells were grown to confluence and infected with either rVACV-mCherry or rVACV-eGFP. After 3 days, cells were harvested by centrifugation and resuspended in 36 mL of TKE buffer (10 mM Tris-Cl, pH 8.0, 10mM KCl, 5 mM EDTA). Resuspended cells were kept on ice for 10 min with periodic gentle vortexing. After this time, 4 mL of 10% Triton X-100 and 100 µL of beta-Mercaptoethanol (BME) was added to the resuspension, followed by another 10-min incubation on ice with periodic gentle vortexing. The nuclei were then pelleted by centrifugation at 1,500 xg for 10 min before the supernatants were spun at 20,000 xg for 30 min to pellet the virus. The virus was then resuspended in 1.6 mL of cold TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) using a 21-gauge needle. After resuspension, the following were added in order with gentle mixing: 2.8 mL of 54% sucrose (w/w), 30 µL BME, 100 µL proteinase-K (10 mg/mL) and the solution was incubated on ice for 15 minutes. Then, 500 µL of 10% SDS was added and the solution was incubated overnight at 37°C. The following day, 1 mL of 4M NaCl was added with gentle mixing so as not to shear the vDNA. vDNA was then extracted three times with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) by first gently mixing for several minutes, followed by a 3-min spin at 4,500 xg to separate the phases. The upper phase was saved after each spin and added to 550 µL of 3M sodium acetate, pH 7.0 after the last spin. Then, 12.5 mL of ice cold 100% ethanol was added with gentle mixing to this solution. The DNA was allowed to precipitate for at least 1 h at -80°C before being pelleted at 4,500 xg for 30 min. The pellet was washed once with 200 µL of 70% ethanol and spun again at 4,500 xg for 10 min. The pellet was allowed to air dry before being resuspended in 100 µL of TE buffer using pipette tips with the ends cut off to avoid DNA shearing. Several freeze/thaw cycles were conducted to ensure complete DNA resuspension before the concentration was determined with a Nanodrop spectrophotometer (ThermoFisher).

Cleavage of vDNA with Cas9 and gRNAs

Streptococcus pyogenes Cas9-fused to a nuclear localization sequence (NLS) was obtained from Macrolab at UC Berkeley. sgRNAs were first assembled as DNA templates by PCR before in vitro transcription with the Megascript T7 RNA polymerase kit according to the manufacturer’s instructions. To assemble the DNA templates, forward and reverse DNA oligonucleotides (oligos) were ordered. The forward DNA oligos contained the T7 promoter (5’-TAATACGACTCACTATAGG-3’), followed by a 20-nucleotide sgRNA sequence (see below for details) and then a sequence to anneal to the 5’ end of the tracrRNA (5’-GTTTTAGAGCTAGAAATAGC-3’). Example forward oligo using the first mCherry guide (underlined) is listed below: 5’-TAATACGACTCACTATAGGTATGCTATAAATGGTGAGCAGTTTTAGAGCTAGAAATAGC-3’. A reverse DNA oligo designed to anneal to the 3’ end of the tracrRNA (5’-AAAAAAGCACCGACTCG-3’) was also ordered. PCR was then conducted using the forward and reverse DNA oligos to amplify each unique guide into a DNA template for in vitro transcription. For the PCR template, a plasmid containing the tracrRNA was used. PCR products were purified with the Qiagen PCR purification kit according to the manufacturer’s instructions before in vitro transcription.

To design sgRNAs, the CRISPOR program was used 19. About 200 bp (NCBI Reference Sequence: NC_006998.1) surrounding the site of the desired insertion were submitted to the program. Potential sgRNAs were then manually mined to minimize off-target effects. sgRNAs that were in close proximity to the desired insertion site with minimal predicted off-targets were chosen. sgRNA sequences are listed in the supplemental Excel file.

For the Cas9 cleavage reaction, two gRNAs were used for each target gene. The Cas9, gRNAs and vDNA substrate were incubated in a 10:10:1 molar ratio with a final Cas9 concentration of 30 nM. The reaction was buffered in 1x New England BioLabs (NEB) buffer 3.1. The reaction was allowed to continue overnight at 37°C before being stopped by heat shock at 65°C for 5 min. To determine the Cas9 cleavage efficiency, 1.5 µg of the Cas9-treated vDNA was digested with either HindIII-HF or XhoI (NEB) for 3 h before being resolved on a large 0.6% agarose gel alongside an untreated control. DNA fragments were visualized with ethidium bromide staining. Throughout this process, cut pipette tips were used to avoid shearing the vDNA.

Generation of transfer amplicons

To generate transfer amplicons for transfection, constructs were first custom-synthesized by either Epoch Life Science or Twist Biosciences and then amplified with primers designed to anneal to the ends of the synthesized sequences. For the eGFP rescue, the eGFP open-reading frame was placed under the control of the p11 promoter (5’-GAATTTCATTTTGTTTTTTTCTATGCTATAAATG-3’) and flanked on either side by 300 bp of homologous sequences at the tk locus. The primers to generate the transfer amplicon are listed in the supplemental Excel file. For A33R and A34R, the desired mutations were synthesized into a gene fragment flanked by about 500 bp of homologous sequences on each side. The specific mutations chosen were as follows; for A33R, the first 142 codons were unchanged, then a 12-nucleotide sequence (5’-TATCTAGCTCAT-3’) replaced the final 43 codons before the stop codon; for A34R, the 151st codon was changed from AAA to GAA. The primers to generate the transfer amplicons for A33R and A34R are listed in the supplemental Excel file. For H2R and L5R, a sequence encoding the 3X-FLAG tag was installed before the stop codon at the C– terminus of each gene and the coding sequence was flanked by 300 bp of homologous sequences on each side. The primers to generate the H2R and L5R are listed in the supplemental Excel file. After PCR of 30 cycles using Phusion polymerase, each amplicon was run on a 1% agarose gel and purified with the Qiagen gel extraction kit.

Rescue of rVACVs

BSC-40 cells were seeded in wells of a 6-well plate at a density of ∼500,000 cells per well. The following day, each well was infected with 1.5 IU/cell of FWPV. Two hours later, 500 ng of cleaved vDNA was co-transfected with 150 ng of an appropriate transfer amplicon using Lipofectamine 3000 (Company) according to the manufacturer’s instructions. After overnight incubation, the media was replaced with DMEM-10% FBS and rVACV production was allowed to occur for 5–7 days. Cell supernatants and pellets were collected by scraping and viruses were released from the cells by three freeze/thaw cycles.

TOPO cloning and sequencing

To estimate the efficiency of homologous recombination between the vDNA and transfer amplicon before proceeding to plaque purification, about 10% of the cell suspension mixed with the cell supernatant was harvested and vDNA was purified with the Qiagen blood mini prep kit according to manufacturer instructions. vDNA was then amplified by PCR with Phusion polymerase. Primers that anneal to the VACV genome outside the transfer amplicon were used to avoid re-amplifying the transfected construct. All “verification” primers used in this study are listed in the supplemental Excel file. After PCR, each amplicon was run on a 1% agarose gel and visualized with ethidium bromide staining.

We next sought to get a better estimation of what percent of the viral genomes had successfully incorporated the mutations for A33R and A34R. To assess this, Phusion polymerase and appropriate verification primers were used to amplify these loci with either 20 or 22 cycles, as determined in the supplemental figure. Three separate PCR reactions were performed for each sample, the products of each reaction were gel extracted and then reactions from the same sample were combined into one tube. The PCR pooled amplicons were then cloned into the pGEM-T plasmid using a TOPO cloning kit (Company) according to the manufacturer’s instructions. Individual bacterial colonies were then subjected to rolling-circle amplification and Sanger sequencing with gene-specific primers.

After purification and amplification of individual viral plaques, vDNA from each plaque was purified with a Qiagen blood mini prep kit. PCR was then conducted with matching verification primers, followed by gel extraction and Sanger sequencing of the PCR products with the same primers.

Plaque assays

After initial virus rescue with the helper virus, individual plaques were grown to assess the efficiency of rVACV formation. After freeze/thawing, a 10-fold dilution series for each rescue was set up in a 6-well plate with a starting dilution of 10−3. One hour after infection, the media was exchanged with a 0.5% methylcellulose overlay and incubated for 2-3 days. Plaques were then randomly selected and the virus was expanded by growing on BSC-40 cells in a 6-well dish. For the assay to determine the size of the A33R plaques, the plaques were grown for 4 days under a 0.5% methylcellulose overlay and then stained with 0.1% crystal violet.

Western blotting

BSC-40 cells were seeded into a 6-well plate and infected with virus. After two days, cells were collected by scraping and the cell pellet was lysed in 1% SDS. Proteins in 20 µg of each lysate were then resolved on a 4–20% polyacrylamide gradient gel and transferred to a PVDF membrane. Membranes were blocked in 4% milk and stained with anti-FLAG (1:2000 dilution) and anti-mouse-HRP (1:5000 dilution) primary and secondary antibodies. Protein bands were detected via chemiluminescence using a Bio-Rad ChemiDoc Touch imager.

Immunofluorescence

BSC-40 cells were seeded onto a coverslip in a 12-well plate and infected with virus. The following day, cells were harvested by fixation for 15 min with 4% paraformaldehyde. Cells were washed 2x with PBS and then permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were washed again with PBS and blocked with 3% BSA in PBS for 10 minutes. A primary anti-FLAG antibody was diluted 1:500 in 3% BSA/PBS and incubated with the coverslip for 1 h. Cells were then washed 5 times with PBS and incubated for 1 h with secondary antibody, which had been diluted 1:1000 in 3% BSA/PBS. Cells were then washed 4 times with PBS, with the penultimate wash containing Hoechst dye at a 1:20,000 dilution. Coverslips were mounted onto slides with Prolong Gold (Company) as a mounting reagent and sealed with nail polish. Images were taken at either 40x or 63x magnification using a Zeiss Axio Observer inverted microscope.

Article TitleMAVERICC: Efficient marker-free rescue of vaccinia virus recombinants by in vitro CRISPR-Cas9 engineering

Abstract

Vaccinia virus (VACV)-based vectors are in extensive use as vaccines and cancer immunotherapies. VACV engineering has traditionally relied on homologous recombination between a parental viral genome and a transgene-bearing transfer plasmid, a highly inefficient process that necessitates the use of a selection or screening marker to isolate recombinants. Recent extensions of this approach have sought to enhance the recovery of transgene-bearing viruses through the use of CRISPR-Cas9 engineering to cleave the viral genome in infected cells. However, these methods do not completely eliminate the generation of WT viral progeny and thus continue to require multiple rounds of viral propagation and plaque purification. Here, we describe MAVERICC (marker-free vaccinia virus engineering of recombinants through in vitro CRISPR/Cas9 cleavage), a new strategy to engineer recombinant VACVs in a manner that overcomes current limitations. MAVERICC also leverages the CRISPR/Cas9 system but requires no markers and yields essentially pure preparations of the desired recombinants in a single step. We used this approach to rapidly introduce point mutations, insertions, and deletions at multiple locations in the VACV genome, both singly and in combination. The efficiency and versatility of MAVERICC make it an ideal choice for generating mutants and mutant libraries at arbitrarily selected locations in the viral genome to build complex VACV vectors, effect vector improvements, and facilitate the study of poxvirus biology.


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