SCC2 Cas9 cell line
The HPV type 16+ OPSCC cell line, UDSCC2 (SCC2), constitutively expressing Cas9 (SCC2 _as9) were developed as previously described 14and maintained in complete media containing DMEM (Gibco-Invitrogen, Waltham, MA) supplemented with 10% heat inactivated foetal bovine serum (FBS) and 1% antibiotic/glutamine preparation (100 U/ml penicillin G, 100 U/ml streptomycin sulphate, and 2.9 mg/ml L-glutamine) (Gibco-Invitrogen, Waltham, MA) and blasticidin (InvivoGen) at 2 mg/ml.
Generating doxycycline inducible CRISPR system in SCC2 Cas9 cells
CRISPick design tool was used to design the gRNAs targeting the HPV 16 E7 and 18 E7 oncogene and cross-checked against other gRNA selection tools (ChopChop and Crispor). The HPV 16 E7 targeting gRNA (5′-GCAAGTGTGACTCTACGCTT-3’) and HPV18 E7 targeting gRNA (5’-CCGGTTGACCTTCTATGTCA-3’) were then cloned into the doxycycline (DOX)-inducible gRNA expression plasmid, FgH1tUTG (Addgene #70183) 20, and confirmed by Sanger sequencing (BigDye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems 2002) before lentivirally infecting SCC2_Cas9 cells. Cells were then purified and sorted on the BD FACSAria™ III Cell Sorter (BD bioscience, San Jose, CA).
Chemicals and animal fodder
To promote sgRNA expression in cell lines, doxycycline hyclate (DOX) (#D9891, Sigma-Aldrich, St Louis, MI) was added to cell culture media at a final concentration of 10µg/ml. Standard rodent food was supplemented with DOX (600mg/kg body weight) (DOX fodder) for in vivo work (#SF08-026, Specialty Feeds, WA, Australia).
Cell viability determination
Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT reagent (Sigma Aldrich, St Louis, MI) was added into cell media at a final concentration of 0.5mg/ml for 2h at 37°C before MTT crystals were dissolved in DMSO to allow measurement of colorimetric absorbance at 544 nm using a FLUOstar OPTIMA microplate reader (BMG LabTech, Germany).
Colony forming assay
Cells were seeded at 50,000 cells per well in a 6-well plate and left overnight to adhere before adding media containing 10µg/ml of dox. DOX-supplemented media was changed daily over seven days before media was removed and cells stained with crystal violet. Plates were visualised and imaged captured on a Chemidoc XRS Visualiser using a white light filter setting (BioRad, Hercules, CA).
SCC2 Cas9 xenografts and DOX treatment
Female nude mice (aged 6-8 weeks) were purchased from the animal resource centre (ARC), Perth, Australia. All animal experiments were performed in accordance with the Australian and New Zealand Council for the Care and Use of Animals in Research standards and were approved by the Griffith University Animal Ethics committee (MHIQ/14/20). Tumours were established by subcutaneously injecting 2×106 respective SCC2_Cas9 cells (100μl/injection in 50% PBS:50% Corning ® Matrigel® solution (vehicle) (Sigma-Aldrich, St Louis, MI) in the right flank of mice as done previously 13. Mice were monitored for tumour growth and when the tumours reached a size of approximately 100 mm3, mice were fed DOX fodder to induce the DOX-inducible CRISPR system as previously described 20. Tumours volumes were measured using digital callipers every two days and mice health monitored daily. Mice were euthanized and culled at the end of designated monitoring period or when tumour mass reached 1000mm3 or reached clinical endpoint (>15% weight loss, lack of grooming, reduce activity and appetite).
Time-lapse microscopy
Cells were followed by time-lapse microscopy using Holometer®, a cell stain-free phase holographic imager (PHI AB, Lund, Sweden) at 37°C and 5% CO2 and data analysed in Hstudio 2.7.5™ (PHI AB, Lund, Sweden) on 24-well plates (STARSTED, Nümbrecht, Germany). Images were captured at 10min intervals.
Immunoblotting
Tumour proteins were extracted using AllPrep DNA/RNA/Protein Kit (#80004, QIAGEN, Hilden, Germany), according to manufacturer’s protocol. Immunoblots were probed with antibodies against HPV 16E7 (NM2) (Santa Cruz Technologies Biotechnologies, Dallas, TX), and S6 (Cell Signaling Technologies, Danvers, MA). Rabbit and mouse secondary antibodies (Cell Signaling Technologies, Danvers, MA) and ECL was used to detect protein signals on a Chemidoc XRS Visualiser (BioRad, Hercules, CA).
Determining gene editing efficiency
Cell genomic DNA was isolated using the QIAamp DNA Mini Kit (#51306 QIAGEN, Hilden, Germany) according to manufacturers’ instructions. Tumour DNA and RNA were extracted using AllPrep DNA/RNA/Protein Kit (# 80004, QIAGEN, Hilden, Germany), according to manufacturers’ protocol. The HPV 16 E7 gene was amplified by PCR using primers previously designed 13 and purified by ultrafiltration before performing Sanger sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems 2002 (Part# 4337035A) following manufacturers’ protocol. Sanger sequencing results were analysed using the online tool, TIDE (Tracking Indels by DEcomposition https://tide.nki.nl) as previously described 21.
Whole exome sequencing and bioinformatics analysis
DNA samples were prepared for sequencing using the Twist Enzymatic Fragmentation (Twist Biosciences) preparation followed by Twist Exome V2 exome capture (Twist Biosciences) at the Australian Genome Research Facility (AGRF). Subsequently, libraries were sequenced on the NovaSeq 6000 (Illumina) at the AGRF using a paired end 150bp sequencing configuration. The FASTQ data were then assessed for quality and contamination before undergoing the somatic variant calling workflow at the AGRF. Briefly, each sample was aligned to the human consensus sequence (hg38) using the DRAGEN Bio-IT platform v3.9.3 (Illumina). Reads identified as PCR or optical duplicates were then marked. Somatic small variant and indel calling was performed using DRAGEN v3.9.3 (Illumina) in a tumour/normal mode with each tumour sample compared against the SCC2 parental cell line. Variant calling was performed within the targeted capture region and included a ‘G/T’ orientation bias filter corresponding to 8-oxoguanine formation caused by oxidation. The resulting variant calls were annotated with the Variant Effect Predictor (VEP) v105.0 22.
Pathway enrichment analysis
Pathway enrichment analysis was performed to identify pathways enriched in the selected gene list. Enrichr 23 was used to perform pathway enrichment analysis, which produces Benjamini-adjusted p-value, z-score, and total enrichment score (which incorporates both p-value and z-score information) for each pathway. Kyoto Encyclopedia of Genes and Genomes (KEGG) was selected as reference pathway database.
Statistical analysis
All statistical analyses were performed using the statistical software package GraphPad Prism v9 and described in detail in respective figure legends.