GFP-on mouse model for interrogation of in vivo gene editing

Carla Dib^#^{1

2}, Jack A Queenan^#^{3

4

5}, Leah Swartzrock^{1

2}, Hana Willner^{1

2}, Morgane Denis^{1

2}, Nouraiz Ahmed^{3

4

5}, Fareha Moulana Zada^{6

7

8}, Beltran Borges^{6

7

8}, Carsten T Charlesworth², Tony Lum^{6

7

8}, Bradley P Yates⁹, Caleb Y Kwon¹⁰, Augustino V Scorzo¹⁰, Scott C Davis¹⁰, Jessie R Davis^{3

4

5}, Ran He^{11

12}, Jun Xie^{11

12

13

14}, Guangping Gao^{11

12

13

14}, Tippi C MacKenzie^{6

7

8}, David R Liu^{15

16

17}, Gregory A Newby^{18

19

20

21

22

23

24}, Agnieszka D Czechowicz^{25

26}

Affiliations

¹ Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
² Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.
³ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁴ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
⁵ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁶ Department of Surgery, University of California San Francisco, San Francisco, CA, USA.
⁷ The Eli and Edythe Broad Center for Regeneration of Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, USA.
⁸ The Center for Maternal-Fetal Precision Medicine, University of California San Francisco, San Francisco, CA, USA.
⁹ Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹⁰ Dartmouth College, Thayer School of Engineering, Hanover, NH, USA.
¹¹ Department of Genetics & Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹² Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹³ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹⁴ Department of Microbiology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹⁵ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹⁶ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹⁷ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹⁸ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA. gnewby@jhmi.edu.
¹⁹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. gnewby@jhmi.edu.
²⁰ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. gnewby@jhmi.edu.
²¹ Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. gnewby@jhmi.edu.
²² Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. gnewby@jhmi.edu.
²³ Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. gnewby@jhmi.edu.
²⁴ Department of Molecular Biology and Genetics, Johns Hopkins University, Baltimore, MD, USA. gnewby@jhmi.edu.
²⁵ Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. aneeshka@stanford.edu.
²⁶ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. aneeshka@stanford.edu.

^# Contributed equally.

PMID: 40744920
PMCID: PMC12313916
DOI: 10.1038/s41467-025-61449-y

GFP-on mouse model for interrogation of in vivo gene editing

Carla Dib et al. Nat Commun. 2025.

. 2025 Jul 31;16(1):7017.

doi: 10.1038/s41467-025-61449-y.

Authors

Affiliations

¹ Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
² Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.
³ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁴ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
⁵ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁶ Department of Surgery, University of California San Francisco, San Francisco, CA, USA.
⁷ The Eli and Edythe Broad Center for Regeneration of Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, USA.
⁸ The Center for Maternal-Fetal Precision Medicine, University of California San Francisco, San Francisco, CA, USA.
⁹ Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹⁰ Dartmouth College, Thayer School of Engineering, Hanover, NH, USA.
¹¹ Department of Genetics & Cellular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹² Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹³ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹⁴ Department of Microbiology, University of Massachusetts Chan Medical School, Worcester, MA, USA.
¹⁵ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹⁶ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹⁷ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
¹⁸ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA. gnewby@jhmi.edu.
¹⁹ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. gnewby@jhmi.edu.
²⁰ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. gnewby@jhmi.edu.
²¹ Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA. gnewby@jhmi.edu.
²² Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. gnewby@jhmi.edu.
²³ Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. gnewby@jhmi.edu.
²⁴ Department of Molecular Biology and Genetics, Johns Hopkins University, Baltimore, MD, USA. gnewby@jhmi.edu.
²⁵ Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. aneeshka@stanford.edu.
²⁶ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. aneeshka@stanford.edu.

^# Contributed equally.

PMID: 40744920
PMCID: PMC12313916
DOI: 10.1038/s41467-025-61449-y

Abstract

Gene editing technologies have revolutionized therapies for numerous genetic diseases. However, in vivo gene editing hinges on identifying efficient delivery vehicles for editing in targeted cell types, a significant hurdle in fully realizing its therapeutic potential. A model system to rapidly evaluate systemic gene editing would advance the field. Here, we develop the GFP-on reporter mouse, which harbors a nonsense mutation in a genomic EGFP sequence correctable by adenine base editor (ABE) among other genome editors. The GFP-on system was validated using single and dual adeno-associated virus (AAV9) encoding ABE8e and sgRNA. Intravenous administration of AAV9-ABE8e-sgRNA into adult GFP-on mice results in EGFP expression consistent with the tropism of AAV9. Intrahepatic delivery of AAV9-ABE8e-sgRNA into GFP-on fetal mice restores EGFP expression in AAV9-targeted organs lasting at least six months post-treatment. The GFP-on model provides an ideal platform for high-throughput evaluation of emerging gene editing tools and delivery modalities.

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Conflict of interest statement

Competing interests: A.C. discloses financial interests in the following entities working in the rare genetic disease space: Beam Therapeutics, Editas Medicines, Fulcrum Therapeutics, Global Blood Therapeutics, Inograft Biotherapeutics, Land Medicine, Prime Medicine, and Spotlight Therapeutics. D.R.L. is a consultant and/or equity holder of Prime Medicine, Beam Therapeutics, Pairwise Plants, and nChroma Bio, companies that use gene editing or genome engineering. J.R.D. is a current employee and equity holder of Prime Medicine. D.R.L. is an inventor of base editors (US 20170121693). D.R.L. and J.R.D. are inventors of AAV vectors encoding base editors (US 20250064981). A.C. and S.C.D. have a financial interest in GV, a venture capital firm funded by Alphabet, and its portfolio. A.C. has received research funding from Rocket Pharmaceuticals, Jasper Therapeutics and STRM.Bio. S.C.D. has received research funding from Aera Therapeutics. None of these entities funded or were involved in this study. The remaining authors report no competing interests.

Figures

**Fig. 1. Generation of GFP-on reporter mouse model.**
a Sequence of the premature termination codon-containing mutant GFP-on allele. Target nucleotide in red converted by CBE to GFP-on allele and restored to WT by ABE. b Schematic detailing the generation of the GFP-on⁻^/pm mouse from the EGFP⁺ male. Created in BioRender. Czechowicz, A. (2025) https://BioRender.com/9ikb7ly. c High-throughput sequencing (HTS) of PCR-amplified genomic DNA extracted from EGFP^pm/pm ear tissue. d Absolute number of EGFP and GAPDH copies quantified by droplet digital PCR. NTC no-template control. e Loss of EGFP fluorescence in GFP-on mice detected after opening the skin of the GFP-on mouse. Left: EGFP⁺ mouse, right: GFP-on mouse. f Loss of GFP expression in peripheral blood, bone marrow, spleen and liver cells in GFP-on mice compared to EGFP⁺ mice. Shown is FACS analysis of EGFP expression. Numbers in green boxes indicate % of live cell population. Source data are provided as a Source Data file.

**Fig. 2. Correction of the EGFP point mutation ex vivo.**
a Schematic of EGFP locus with three guide RNAs used to correct the mutation to restore EGFP expression. b Highest editing efficiency observed with sgRNA1. Data indicated the percentage of EGFP correction in EGFP^pm/pm fibroblasts electroporated with SpABE8e mRNA. n = 2 samples, technical replicates. Data are presented as mean with Standard Deviation (SD). c SpABE8e mRNA and sgRNA1 electroporation restores EGFP expression in c-Kit⁺ bone marrow cells. Shown are FACS plots of EGFP expression in c-Kit+ bone marrow cells with or without SpABE8e mRNA electroporation. Numbers in the green plots indicate % of live cells. EGFP restoration in EGFP^pm/pm fibroblasts transduced with dual AAV9 (n = 3, technical replicates) assessed by d fluorescence microscopy (×10 magnification, 130 µm), e FACS, and f HTS. Source data are provided as a Source Data file.

**Fig. 3. Correction of the EGFP point mutation in adult mice.**
Assessment of EGFP expression in various organs of GFP-on^pm/pm mice (n = 3, biological replicates) 3 weeks post systemic in vivo treatment with dual AAV9 containing SpABE8e-sgRNA1 via a flow cytometry, b fluorescence microscopy (×10 magnification, 100 µm), and c HTS. Data are presented as mean. PB peripheral blood, BM bone marrow. Source data are provided as a Source Data file.

**Fig. 4. Correction of the EGFP point mutation in fetal mice.**
Assessment of EGFP expression in various organs of GFP-on^pm/pm mice (n = 3, biological replicates) post-treatment in utero with dual AAV9 containing SpABE8e-sgRNA1 at a 8–12 weeks post-birth in various organs via a flow cytometry, b fluorescence microscopy (×10 magnification, 100 µm), and c HTS. Data are presented as mean. PB peripheral blood, BM bone marrow. Source data are provided as a Source Data file.

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References

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GFP-on mouse model for interrogation of in vivo gene editing

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GFP-on mouse model for interrogation of in vivo gene editing

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