Abstract
Base and prime editing technologies precisely install defined nucleotide edits in both dividing and non-dividing cells, offering potential for correcting pathogenic mutations directly in organisms. However, to fully leverage their therapeutic potential, accurately measuring editing rates with high spatial resolution is crucial. Here we use imaging-based in situ sequencing (ISS) to map base and prime editing events within native tissues. We establish and validate this technology in mouse brains treated with intein-split adenine base editors or prime editors delivered via adeno-associated viral vectors. We next apply ISS in the liver of mice and macaques treated with adenine base editors encoded on lipid nanoparticle-encapsulated mRNA and guide RNA (RNA-LNP). Effective editing was observed across all metabolic zones of liver lobules. Moreover, in experiments where repeated doses of RNA-LNP are administered, the initial dose does not affect the editing efficiency and distribution of the subsequent dose. Our results demonstrate how ISS can visualize gene editing events in vivo and suggest that base editor delivery using RNA-LNP could be used to address a wide spectrum of metabolic liver diseases.
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Data availability
All oligonucleotides and padlock probes used in the study are provided in Supplementary Table. HTS data have been deposited at the Sequence Read Archive (PRJNA1102160)49. Source data are provided with this paper.
Code availability
The CellProfiler pipeline and R script is available via GitHub at https://github.com/sharan-j/genome_editing_ISS (ref. 50).
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Acknowledgements
We thank the Functional Genomics Center Zurich for technical support and access to instruments at the University of Zurich; and members of the laboratory of G.S. and Acuitas for discussions and comments on the paper. This work was supported by the European Molecular Biology Organization long-term fellowship EMBO ALTF 873-2019 (to S.J.), a Novartis Young Investigator Grant (to S.J.), Swiss National Science Foundation grant number 310030_185293 (to G.S.), a European Research Council Consolidator Grant (to G.S.) and the Helmut Horten Foundation (to G.S.). The Pardi Laboratory was supported by the National Institute of Allergy and Infectious Disease of the US National Institutes of Health under award numbers R01AI153064, P01AI158571 and P01AI172531.
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G.S., S.J., S.C.S. and Y.K.T. conceived the study. S.J. and T.H. set up and performed all ISS experiments and analysis. T.R., L.K., M.W. and D.B. performed in vivo injections into mice. N.M. and K.M. performed experiments on GFP reporter cells. W.J.M. performed RNA-LNP formulations; H.M., M.V. and N.P. performed the mRNA production. T.C.C., S.C.S., Y.K.T., S.J., T.H. and G.S. analysed data from the macaque experiments. G.S. and S.J. wrote and edited the paper with input from all co-authors. All authors read and approved the final version of the paper.
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T.C.C., W.J.M., S.C.S. and Y.K.T. are employees of Acuitas Therapeutics. G.S. and K.M. are co-founders of Nerai Bio. G.S. is a scientific adviser to Prime Medicine. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Detection of prime editing by ISS in cultured cells.
(a) Schematic of the experimental setup to detect prime editing in GFP reporter cells treated with PE2. (b) Quantification of the percentage of edited reads using NGS. Each datapoint represents an independent well of separately transfected cells. (c) Image shows edited (green) and unedited GFP reporter cells (left panel), and RCPs (right panel). Scale bar: 10 um. (d) Quantification of the fraction of GFP positive cells and edited reads detected using ISS. Each point represents an independent well (replicate 1, n = 805 cells; replicate 2 = 704 cells). (e) Quantification of the concordance between GFP expression and ISS in prime edited GFP reporter cells.
Extended Data Fig. 2 Detection of base editing by ISS in the mouse brain after intracranial AAV injection.
(a) (Left) Schematic of the experimental set-up. 2 μl AAV PHP.eB with a concentration of 1×1013 vg/mL encoding for the ActbV30 targeting ABEmax were injected into the hypothalamus of adult mice (Right). Image from the Allen Brain Atlas. Depicting areas illustrate the areas analyzed by NGS (yellow) or ISS (red). (b) Representative brain tissue region cut by laser microdissection from mice injected in hypothalamus. (c) Quantification of editing efficiencies in the hypothalamus of treated mice detected by NGS and ISS. Datapoints represent different mice. n = 3922 and 1703 RCPs. (d) Image shows RCPs in the hypothalamus of mice treated with ABE targeting ActbV30. Insets show the RCPs as single channel images. Scale bar: 10 um. (e) Quantification of editing rates in distinct sub-regions of the hypothalamus. Datapoints represent separate areas in the hypothalamus from at least two mice. Left: n = 521 and 659 RCPs; mid: n = 1623 and 807 RCPs; right: n = 1778 and 237 RCPs. Bars represent mean ± s.d. a, Created with BioRender.com.
Extended Data Fig. 3 Biodistribution of AAV9 and RNA-LNP in mice liver.
(a) (Top) Schematic of the experimental set-up. Adult mice were injected via the tail-vein with either AAV9 TagRFP-expressing vector or LNP-mCherry. (Bottom) Representative images of the liver lobule zonation from treated mice. Pericentral cells are counterstained with Glutamine Synthetase (GS) (magenta) antibody and periportal cells are counterstained with ECAD (green) antibody. Scale bar: 100 um. (b) Quantification of average intensities in arbitrary units of either TagRFP or mCherry in the three liver zones. Datapoints represent independently treated animals. a, Created with BioRender.com.
Extended Data Fig. 4 Analysis of editing in extrahepatic tissues liver and toxicity markers after systemic RNA-LNP delivery into macaques.
Quantification of (a) liver toxicity markers and (b) inflammatory biomarkers or cytokines before and during treatment. Bars represent mean ± s.d. Empty datapoints represent animals from strategy 1 and filled datapoints represent animals from strategy 2. Circle and square datapoints represent values of independently treated animals (2 animals per strategy). Vertical dotted lines represent the time point of I.V. infusion. (c-d) Quantification of editing in extrahepatic organs in animals from strategy 1 and 2. Each datapoint represents the values of independently treated animals. A control group was not included due to the unavailability of liver tissue from untreated macaques.
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Oligonucleotide and padlock probe sequences used in the paper, and percentage editing rates from NGS and ISS.
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Janjuha, S., Haenggi, T., Chamberlain, T.C. et al. Spatial profiling of gene editing by in situ sequencing in mice and macaques. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01512-7
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DOI: https://doi.org/10.1038/s41551-025-01512-7
