Fig 1.
Schematic overview of the experimental design.
Hamsters were infected with juvenile Opisthorchis viverrini worms that had been subjected to gene knockout. Two experiments were conducted. (A). The goal of experiment 1 was to assess the impact of CRISPR/Cas9 editing on fluke survival. The goal of experiment 2 was to assess the influence of gene knockout on pathogenesis and malignant transformation. Groups of hamsters were infected with flukes transfected with CRISPR/Cas9 plasmids targeting either Ov-grn-1 (red, ΔOv-grn-1), Ov-tsp-2 (green, ΔOv-tsp-2), or irrelevant guide RNA (blue, control) and exposed to dimethyl nitrosamine (DMN) in the drinking water. At timepoints indicated for each experiment, fecal egg numbers were measured as eggs per gram of feces (EPG). Experiment 1: From the livers of euthanized hamsters, all the flukes were recovered and transcript levels of the targeted genes assessed. Experiment 2: Liver lobes were sectioned and stained for histochemical analysis. Flukes incidentally released from bile ducts were collected and assessed for gene knockout. Before infection, transcript levels of Ov-grn-1 (B) and Ov-tsp-2 (C) were assessed in juvenile flukes. Transcript levels established by qPCR were plotted relative to average control transcript levels from 2–4 biological replicates; average shown with colored bar and with 95% confidence interval bars. Population statistics were generated from resampling 1000 times by replacement bootstrap analysis of untransformed delta-delta Ct values (derived from S1 Fig). (Elements of the figure include icons from BioRender.com with copyright and licensing permission).
Fig 2.
Liver fluke burden and levels of gene transcription.
Fecundity, worm numbers, and gene expression levels were determined at 10–14 weeks after infection of the hamsters with 100 gene edited juveniles, and from three hamsters per group (Experiment 1). Number of eggs per gram of feces (EPG) from each hamster at weeks 10 and 12 (A) and worm numbers at week 14 (B) showing mean (horizontal black line) and SEM bars. Each treatment group was compared to the control group in 2-way ANOVA with Holm-Sidak multiple comparison: ns = not significant; **, P ≤ 0.01; ***, P ≤ 0.001, and ΔOv-grn-1 against ΔOv-tsp-2: #, P ≤ 0.05. Gene transcript levels of Ov-grn-1 (C) and Ov-tsp-2 (D) were determined by qPCR for 10 to 13 flukes sampled from each animal (30–39 flukes total per group) and plotted with each datum point representing the transcript level of an individual fluke relative to wild-type flukes. Resampling with replacement bootstrap analysis (B = 1000) of ddCT scores (S3 Fig) was used to generate population average, as denoted by the thick, colored line and 95% confidence interval bars.
Fig 3.
Cholangiocarcinoma model, fecundity, gene transcript and mutation rates.
Eggs per gram of feces (EPG) were assessed at week 23 prior to euthanasia at week 24 (Experiment 2). Panel A, EPG values of the three groups of hamsters. Violin plot denotes each hamster’s EPG with “x” symbols. Solid colored lines indicate the median values and dashed black lines indicate the quartiles. Kruskal Wallis with Dunn’s multiple comparison correction was used to compare EPG levels against control group: *, P ≤ 0.05; ns, not significant. At necropsy, a sample of 12 to 20 flukes were collected from each group, transcript levels determined for Ov-grn-1 (B) and Ov-tsp-2 (C), and plotted with each datum point representing the transcript level of the individual fluke relative to wild-type flukes. Resampling with replacement bootstrap analysis (B = 1000) of ddCT scores (derived from S4 Fig) was used to generate population average–denoted by the thick, colored line and 95% confidence interval bars.
Fig 4.
Gene mutation rates among liver flukes.
Panel A, Programmed gene knock-out was highly efficient—although not in every fluke. The percentage of total indels (insertions/deletions) determined by next-generation sequencing was plotted on the vertical axis of juvenile and 24 wk adult flukes from Experiment 2. Control group juvenile (NEJ) and adult flukes were each from a single pooled sample while juvenile ΔOv-grn-1 were from pools of two biological replicates, and ΔOv-grn-1 adults from 13 individual flukes. The highly edited flukes were denoted HΔOv-grn-1, flukes with low editing denoted LΔOv-grn-1, and the single fluke with median level editing denoted MΔOv-grn-1. One sample t-test for either juvenile or adult worms comparing ΔOv-grn-1 and control: *, P ≤ 0.05; **, P ≤ 0.01. The thick solid line is the median and black dashed lines represent the inter-quartiles. A broken Y-axis with a magnified lower portion highlights the near zero values. B. Adult fluke indel mutation rate was inversely correlated with transcript level. The indel and transcript levels were plotted for each individual ΔOv-grn-1 fluke (red circles, combining data from Figs 3C and 4A). Two-tailed non-parametric correlation determined by Spearman co-efficient: **, P ≤ 0.01. For context, the control indel percentages were plotted against the transcript median (blue triangle) with interquartile range error bars. C. ΔOv-grn-1 indel location and size. The NGS reads revealed distinct indel patterns in 12 of 13 adult flukes. Shown as a multivariate bubble plot, the amplicon base pair open reading frame (ORF position) was plotted against the average indel length. The diameter of the bubbles (1–11) reflected how many of 13 adult flukes recorded a matching indel. The programmed double stranded break between residues 19 and 20 was indicated on the X-axis by the term “cut”. For clarity, deletions in adult worm genomes (blue) and insertions (red) have been nudged up/down on the y-axis ±0.1. The deletions in juvenile worms (yellow) are shown from one pooled sample. Insertions were not seen. Position -9 was highlighted with a vertical dotted line and the black horizontal square bracket (└─┘) highlighted a cluster of mutations. The sequence around this cluster was shown below the x-axis and the initiator ATG codon indicated in red. D. Mutation rate (indels) at each location: the graph plotted the nucleotide position against the percentage frequency of indels in individual flukes. A vertical dotted line highlighted a mutational hotspot at nucleotide -9 and the black horizontal bracket (└─┘) marked a mutational cluster. Other indels of note were labeled with the nucleotide and position.
Fig 5.
Burden of disease in liver fluke infection-associated cholangiocarcinoma.
After resection of the livers at necropsy, a fragment of each lobe was either fixed in formalin for downstream thin sectioning or was manually disrupted to release flukes, which in turn were examined for gene editing events (Fig 1, Experiment 2). Gross anatomical appearance and histopathological results during induction of cholangiocarcinoma [31]. Multiple CCA nodules in the hamster liver were present on both diaphragmatic (A) and visceral surfaces (B). Micrographs of H&E-stained thin sections of liver highlighting foci of moderate dysplasia (C). This image shows bile ducts (blue #) encircled by dysplastic biliary epithelium (yellow arrow) surrounded by fibrosis (fb) with hepatocytes (h) to the left. H&E- stained images of CCA from each of the groups of hamsters group: control (D), ΔOv-grn-1 (E), and ΔOv-tsp-2 (F). Inflammation marked with green asterisk (*), cholangiocarcinoma labeled as CCA; other labels as in panel C. G. Assessment and scoring of lesions was undertaken independently by two co-authors (both veterinary pathologists) using anonymously labeled (blinded) micrographs. The severity of lesions increased from normal tissue (grey) to high grade CCA spanning multiple liver lobes (red). H. EPG from individual hamsters plotted against disease burden on a scale of zero (0, no lesion) to 6 (high CCA) scale. Data plots were slightly reformatted (nudged ± 0.1 on Y-axis) to enable display of overlapping points. Linear regression lines (which were not statistically significant) are shown in shaded color with 95% confidence intervals.
Fig 6.
Attenuated liver fibrosis during infection with ΔOv-grn-1 knockout parasites.
Panel A. Representative images of hepatic fibrosis stained by Picro-Sirius Red with CRISPR-Cas9 edited O. viverrini. Fibrosis was denoted as pink/red thick bands around the bile ducts (periductal fibrosis, fb) and expanded from each portal triad with fibrous septa. OV = Opisthorchis viverrini, H = hepatocyte, BD = bile duct, BE = biliary epithelium. B. Global liver fibrosis: Livers were scored for fibrosis using an Ishak Stage grading scale and plotted on the scale spanning from zero (no fibrosis) to six (cirrhosis) (n = 14 to 17 liver lobes per group).). C. Fibrosis proximal to flukes: Automated ImageJ fibrosis evaluation of the percentage of collagen deposition in images surrounding fluke-containing bile ducts (n = 46 to 54 images per group). Panels B, C: medians shown as thick colored line and dashed black lines mark the inter-quartile ranges. Kruskal-Wallis test with Dunn’s multiple comparisons: against the control group, ns = not significant; *, P ≤ 0.05; ***, P ≤ 0.001, and against the ΔOv-tsp-2 group: #, P ≤ 0.05; ##, P ≤ 0.01.
Fig 7.
Reduced proliferation and minimal mutant p53 expression in cholangiocytes in hamsters infected with ΔOv-grn-1 genotype liver flukes.
Representative images of biliary cells that incorporated BrdU from regions proximal to flukes in control, ΔOv-grn-1 and ΔOv-tsp-2 groups (A). The boxed region in the upper image is expanded in the lower panel. The brown arrow highlights the positive BrdU-stained nuclei and the blue arrow highlights a bile duct cell that did not incorporate BrdU. BrdU index measured from cholangiocytes adjacent to where a fluke was located (n = 27 to 42 per group). (B). Representative micrograph of p53 immunohistochemical staining of biliary epithelium during infection with gene edited flukes (C). Anti-mutant p53 antibody stained the nuclei brown (brown arrows); blue arrows indicate negative cells. Black dashed box in upper wide-angle image magnified in the lower image to aid visualization. Positivity rate (percentages) of mutant p53-positive cholangiocytes (n = 29 to 39 images per group) (D). Where available, 500 to 800 cells were scored from sections of each of the left, middle, and right lobes of the liver marked by “X”. Fewer cholangiocytes (300–500) were available for assessment in several samples, denoted byⓧ Panels A and C: OV = Opisthorchis viverrini, H = hepatocytes, BD = bile duct, BE = biliary epithelium. Panels B, D: non-parametric Kruskal-Wallis test with Dunn’s multiple comparison correction compared against control: ns = not significant; ***, P ≤ 0.001, or against ΔOv-tsp-2: #, P ≤ 0.05; ###, P ≤ 0.001. Thick colored lines signify the median and the dashed black lines denote the inter-quartile range.
Table 1.
Criteria for histopathological and histochemical assessment and grading.