TIGER: A tdTomato in-vivo genome-editing reporter mouse for investigating precision-editor delivery approaches
Data files
Sep 03, 2025 version files 79.29 KB
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Figure_1.xlsx
9.52 KB
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Figure_2.xlsx
9.68 KB
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Figure_3.xlsx
11.70 KB
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Figure_4.xlsx
10.32 KB
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README.md
3.33 KB
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Supplemental_Figure_1.xlsx
14.85 KB
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Supplemental_Figure_2.xlsx
10.78 KB
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Supplemental_Figure_3.xlsx
9.12 KB
Abstract
In vivo genome editing has the potential to address many inherited and environmental disorders. However, a major hurdle for the clinical translation of genome editing is safe, efficient delivery to disease-relevant tissues. A modality-agnostic reporter animal model that facilitates rapid, precise, and quantifiable assessment of functional delivery and editing could greatly enhance the evaluation and translation of delivery technologies. Here, we present the development of the TIGER (tdTomato in vivo genome-editing reporter) mouse, a reporter strain that harbors an integrated and constitutively expressed mutated tdTomato gene in the Polr2a locus. The mutations (Q115X, Q357X) abolish fluorescence, but successful adenine base editing (ABE) or prime editing (PE) restores tdTomato fluorescence. This mouse model facilitates the tissue- and cell-type-specific assessment of genome editing agent delivery. We describe several editing strategies validated in vitro and demonstrate efficient ABE and PE in vivo using viral and non-viral delivery vectors targeting four cell types within the mouse eye: the retinal pigment epithelium (RPE), photoreceptors, Müller glia, and the trabecular meshwork. We show direct editing characterization in the ocular tissues via in vivo and ex vivo two-photon confocal microscopy, and verify the spectral and fluorescence lifetime properties of tdTomato reporter in other mouse tissues. Additionally, we demonstrate successful AAV-mediated prime editing of extraocular tissues, including hepatocytes, skeletal muscle, and brain neurons, by intravenous injection. Thus, the TIGER mouse facilitates the direct development, comparison, and optimization of delivery platforms for efficient and productive ABE or PE broadly applicable in vivo across multiple tissues tested in this study.
Dataset DOI: 10.5061/dryad.866t1g23p
Description of the data and file structure
In vitro and in vivo evaluation of base and prime editing approaches for the characterization of a new tdTomato gene editing reporter mouse.
Files and variables
- Figure_1.xlsx
- 1E: treatment vs. NGS reads with indicated edit (%)
- A7: only target adenine (position 7 in protospacer) edited
- A7+ A9: target adenine and bystander adenine (positions 7 and 9 in protospacer) edited
- Indel: insertions/deletions (unintended edits)
- 1E: treatment vs. NGS reads with indicated edit (%)
- Figure_2.xlsx
- 2E: tissue vs. ln(CPM) from scRNAseq (AU)
- Normalized count per million bp (CPM) from single cell RNA sequencing
- 2E: tissue vs. ln(CPM) from scRNAseq (AU)
- Figure_3.xlsx
- 3B: treatment vs. wavelength (nm) vs. fluorescence (AU)
- eVLP: engineered virus-like particle delivering base editor protein
- 3E: tissue location vs. wavelength (nm) vs. fluorescence (AU)
- RPE: retinal pigment epithelium (retina)
- ONL: outer nuclear layer (retina)
- 3B: treatment vs. wavelength (nm) vs. fluorescence (AU)
- Figure_4.xlsx
- 4F: treatment vs. NGS reads with indicated edit (%)
- Indel: insertions/deletions (unintended edits)
- 4H: tissue location vs. tdTomato positive cells (%)
- PR: photoreceptors
- MG: Muller glia
- AAV8: dual adeno-associated virus serotype 8 delivering prime editor as DNA
- ENVLPE: virus-like particle delivering prime editor protein
- 4F: treatment vs. NGS reads with indicated edit (%)
- Supplemental_Figure_1.xlsx
- SF1A: treatment vs. tdTomato positive cells by flow cytometry (%)
- WT: wildtype tdTomato
- STOP: mutated tdTomato
- STOP + 8e + A6: mutated tdTomato plus ABE8e plus A6 sgRNA
- STOP + 8e + A7: mutated tdTomato plus ABE8e plus A7 sgRNA
- SF1B: treatment (multiple different ABE variants) vs. tdTomato positive cells by flow cytometry (%)
- SF1C: treatment vs. tdTomato positive cells by flow cytometry (%)
- SF1D: different pegRNAs vs. NGS reads with indicated edit (%)
- SF1E: pegRNA vs. NGS reads with indicated edit (%)
- PRIDICT: TIGER correcting pegRNA
- peg12: TIGER correcting pegRNA
- n1-4: TIGER nicking sgRNA for PE3 prime editing strategy
- SF1F: pegRNA vs. NGS reads with indicated edit (%)
- PRIDICT: TIGER correcting pegRNA
- peg12: TIGER correcting pegRNA
- n1-4: TIGER nicking sgRNA for PE3 prime editing strategy
- SF1A: treatment vs. tdTomato positive cells by flow cytometry (%)
- Supplemental_Figure_2.xlsx
- SF2B: treatment vs. tdTomato positive cells by flow cytometry (%)
- Negative: no tdTomato fluorescence
- Medium: medium tdTomato fluorescence
- High: high tdTomato fluorescence
- SF2D: treatment vs. NGS reads with indicated edit (%)
- tt: mutant/mutant tdTomato
- tT: mutant/WT tdTomato
- TT: WT/WT tdTomato
- RNP: ABE8e-N108Q delivered as protein complex
- VLP: ABE8e-N108Q delivered as PE protein by virus-like particle
- SF2B: treatment vs. tdTomato positive cells by flow cytometry (%)
- Supplemental_Figure_3.xlsx
- SF3B: treatment vs. NGS reads with indicated edit (%)
- A7: only target adenine (position 7 in protospacer) edited
- A9: only bystander adenine (position 9 in protospacer) edited
- A7+ A9: target adenine and bystander adenine (positions 7 and 9 in protospacer) edited
- total A7: all reads where target adenine A7 was edited
- SF3B: treatment vs. NGS reads with indicated edit (%)
Code/software
.xlsx: Microsoft Excel, Pages, OpenOffice