Plasmid safe

Pseudovirus (PsV) and Quasivirus (QV) particles were produced by transfecting 293TT cells with HPV16 sheLL plasmids (a gift from the Schiller Lab, NCI, Bethesda, MD, USA) together with either the plasmid for secreted alkaline phosphatase (pSEAP) or cottontail rabbit papillomavirus (CRPV) genome respectively as previously described [23 (link),24 (link),45 (link),46 (link),47 (link)]. Two days post-transfection, cells were collected and lysed using the detergent Brij58 (Sigma-Aldrich, St. Louis, MO, USA). Particles were matured by incubating cell lysates at 37 °C for 24 h and subsequently treating with Benzonase (Sigma-Aldrich, St. Louis, MO, USA) and Plasmid Safe (Epicentre, Madison, WI, USA). Purification of the resulting particles was performed by ultracentrifugation of the lysates at 234,000× g for 3.5 h on an Optiprep step gradient (Sigma-Aldrich, St. Louis, MO, USA). Particles were harvested by puncturing the bottom of the centrifuge tube and collecting fractions of ~250 µL.

HPV16 PsVs were produced as described by Buck and colleagues (standard protocol) [14 (link), 36 ]. 293TT cells were transfected with p16sheLL (expressing L1 and L2 proteins) and pYSEAP (expressing SEAP) using Lipofectamine 2000 (Invitrogen, USA). The transfected cells were cultured for 72 h then harvested and lysed. The plasmids were kindly provided by Dr. J. T. Schiller (NIH, Bethesda, USA). To prepare a mature PsV stock, the lysate was treated with 0.1 % Benzonase (Sigma, USA) and 0.1 % Plasmid Safe (Epicentre, USA) DNase, and incubated for 24 h at 37 °C. The NaCl concentration of the lysate was adjusted to 0.8 M and clarified by centrifugation at 12,000 × g for 10 min at 4 °C. The PsVs in the clarified lysate were purified by SEC as follows: the clarified lysate (0.5 mL) was loaded onto a column (Tricon 10/300, 1 × 32 cm, GE Healthcare, USA) packed with Superose-6 resin (GE Healthcare, USA). The column was equilibrated with working buffer [phosphate buffered saline (PBS) + 0.52 M NaCl + 0.01 % Tween 80 pH 7.2, final NaCl concentration 0.65 M] prior to loading the sample, and the SEC was performed at a flow rate of 0.3 ml/min. Twenty fractions (0.9 mL each) were collected and analyzed by SDS-PAGE and Western blotting.

Single pGG vectors were generated using the same oligonucleotide ligation approach described above. Primer sequences can be found in S1 Table. Arrays were then generated using golden gate assembly. Briefly, 150 ng of each pGG-gRNA plasmid was combined with 150 ng of the appropriate pACPT vector, followed by addition of 1 uL of BsaI (New England BioLabs), 1 uL of T4 DNA Ligase and Buffer (New England BioLabs) and water to a total of 20 uL. Golden gate assembly was then carried out using the following thermocycling protocol: (37°C 5 minutes, 16°C 10 minutes) x10, 50°C 5 minutes, 80°C for 5 minutes and then cooled to 4°C. One uL of 25 mM ATP and 1 uL of Plasmid Safe (Epicentre) were then added to the reaction and incubated for 1 hour at 37°C. Assembled plasmids were then transformed into TOP10 bacteria (Thermo Fisher Scientific) and plated on spectinomycin selection plates with X-gal and mini-preps performed on white colonies using the GeneJET Plasmid Miniprep Kit (Life Technologies). The plasmids were then analyzed by Sanger sequencing to confirm proper gRNA array assembly.