DOI: 10.5061/dryad.866t1g23p
title: TIGER: A tdTomato in-vivo genome-editing reporter mouse for investigating precision-editor delivery approaches

description: 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.
description: # TIGER: A tdTomato in-vivo genome-editing reporter mouse for
 investigating precision-editor delivery approaches Dataset DOI:
 [10.5061/dryad.866t1g23p](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) * Figure_2.xlsx * 2E: tissue vs. ln(CPM) from scRNAseq
 (AU) * Normalized count per million bp (CPM) from single cell RNA
 sequencing * 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) * 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 *
 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 *
 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 * 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 ## Code/software .xlsx: Microsoft Excel,
 Pages, OpenOffice