Fig. 3: In vivo gene editing demonstrated by liver LNP-crRNA targeting and AAV-mediated tumourigenesis.

a, Schematic showing the packaging and delivery of crRNA using LNP to knockout Ttr gene in the liver. Non-targeting control crRNA 1 (NTC1) were packaged as control in the same batch. Constitutive enAsCas12a-HF1 mice were intravenously injected with LNP-crRNA. Serum samples were collected for ELISA to measure and monitor the TTR protein level in serum. Created in BioRender. Chen, S. (2025) https://BioRender.com/j37t723. b, Serum TTR level (µg ml⁻¹) from samples collected at day 0 (before injection), day 6, day 12 and day 20 post injection, via retro-orbital blood draw. The serum TTR level was measured by ELISA. Two independent guides (TTRcr1 and TTRcr2) targeting murine Ttr gene were compared with non-targeting control crRNA (NTC1). Two-way ANOVA with Dunnett’s multiple comparisons test was used to assess significance. Data are shown as mean ± s.e.m. P values are labelled. For all groups, N = 5 biological replicates. c, Pairwise co-occurrence (CO) and mutual exclusivity (ME) analysis for TSGs: TP53, APC, PTEN, RB1, SMAD4 and STK11 on human cancer data from MSK-IMPACT project. Positive log₂(odds ratio) indicated CO, while negative log₂(odds ratio) indicated ME^40,41. We identified TP53, APC, PTEN and RB1 as a group of co-occur genes out of the six TSGs (surrounded by blue lines). SMAD4 was excluded owing to ME with PTEN, RB1 and STK11. STK11 was excluded because it was mutually exclusive with PTEN and SMAD4. d, Schematic showing the core segment of the AAV construct used for tumour induction, which contained a crRNA expression array (crTSG) that included four guides targeting Trp53, Pten, Apc and Rb1, and a Cre expression cassette for inducing enAsCas12a-HF1 expression. After production and purification, AAV-crTSG and AAV-vector (as the negative control) were either intravenously or intratracheally injected into LSL-enAsCas12a-HF1 mice. Tumour and major organs were isolated for NGS and histology analysis. Partial element created in BioRender. Chen, S. (2025) https://BioRender.com/l72d475. e, Representative IVIS spectrum images detecting GFP signal indicated by total radiance (p s⁻¹ cm⁻² sr⁻¹) for tumour and major organs. Top row being representative AAV-crTSG intravenously injected mouse and the bottom row being representative AAV-vector intravenously injected mouse. f, Representative H&E staining and IHC staining on AAV-induced salivary gland SCC FFPE samples. For IHC, tumour samples were stained with GFP (enAsCas12a-HF1-eGFP) and Ki67 (proliferation marker). Black triangles point out the representative GFP⁺ cancerous cell. For 10× images, scale bars = 300 µm. For 40× images, scale bars = 60 µm. g, Representative H&E staining and IHC staining on AAV-induced lung adenocarcinoma FFPE samples. For IHC, tumour samples were stained with GFP (enAsCas12a-HF1-eGFP). Black triangles point out the representative GFP⁺ cancerous cell. For 10× images, scale bars = 300 µm. For 40× images, scale bars = 60 µm. h, Histology section used for CODEX. Top: global view H&E staining on the AAV-induced salivary gland SCC FFPE sample. Clusters 1, 2, 4 and 10 from unsupervised clustering in the UMAP plot in i were labelled with black box. Scale bar, 1 mm. Bottom: global view GFP staining on the same SCC FFPE sample. Scale bar, 1 mm. i, CODEX results reveal the heterogeneity in the tumour samples. Right: UMAP dimensional reduction plot showing the unsupervised clustering of different cell types in the SCC FFPE sample. Left: spatial display of the 17 cell types overlaying the H&E image. Scale bar, 1 mm. j, Heatmap showing the differential expression of the 22 CODEX markers in the 17 cell-type clusters. The expression level was shown as Z score.

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