Figure 8.

Quantification of HBV and SaCas9 RNA levels

(A) Liver sections from three AAV-SaCas9 control and three AAV-SaCas9-treated humanized liver FRG mice were subjected to RNAscope using custom HBV and SaCas9-specific probes. Cells in each image were assigned based on DAPI staining, and those containing foci indicative of HBV and/or SaCas9 probe hybridization were quantified using a RRScell image analysis algorithm, which was used to assign individual cell types. (B) Examples of assigned HBV⁺/SaCas9⁺ cells (red circles), HBV⁺/SaCas9⁻ cells (green circles), HBV⁻/SaCas9⁺ cells (blue circles), and HBV-/SaCas9- cells (purple circles) present within the boxed region of mouse HBV-L1 shown in (A). (C) where appropriate, semi-automated image masking by RRScell was used to analyze non-tumor regions only (yellow areas). (D–F) Quantification of the percentage of HBV⁺ cells per DAPI⁺ cell (D), SaCas9⁺ cells per HBV⁺ cell (E), and HBV⁺ cells per SaCas9⁺ cell (F) is shown for individual humanized mouse livers. Tumors (T) and approximate tumor margins (dotted lines) are indicated where present.