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

Carla Dib; Jack A Queenan; Leah Swartzrock; Hana Willner; Morgane Denis; Nouraiz Ahmed; Fareha Moulana Zada; Beltran Borges; Carsten T Charlesworth; Tony Lum; Bradley P Yates; Caleb Y Kwon; Augustino V Scorzo; Scott C Davis; Jessie R Davis; Ran He; Jun Xie; Guangping Gao; Tippi C MacKenzie; David R Liu; Gregory A Newby; Agnieszka D Czechowicz

doi:10.1038/s41467-025-61449-y

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

Nat Commun. 2025 Jul 31;16(1):7017. doi: 10.1038/s41467-025-61449-y.

Authors

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.

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.

MeSH terms

Animals
CRISPR-Cas Systems
Codon, Nonsense
Dependovirus / genetics
Female
Gene Editing* / methods
Genes, Reporter
Genetic Vectors / genetics
Green Fluorescent Proteins* / genetics
Green Fluorescent Proteins* / metabolism
Humans
Mice
RNA, Guide, CRISPR-Cas Systems / genetics

Substances

Green Fluorescent Proteins
enhanced green fluorescent protein
RNA, Guide, CRISPR-Cas Systems
Codon, Nonsense

Abstract

MeSH terms

Substances

Grants and funding