Abstract
The use of prime editing—a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks—to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. The deep sequencing data generated for this study are available from the NCBI Sequence Read Archive under accession numbers SRR12778000 and PRJNA732214. Source data are provided with this paper.
References
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat. Biotechnol. 39, 198–206 (2021).
Schene, I. F. et al. Prime editing for functional repair in patient-derived disease models. Nat. Commun. 11, 5352 (2020).
Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27 (2020).
Paulk, N. K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010).
Aponte, J. L. et al. Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc. Natl Acad. Sci. USA 98, 641–645 (2001).
Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).
Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016).
Shin, J. H., Jung, S., Ramakrishna, S., Kim, H. H. & Lee, J. In vivo gene correction with targeted sequence substitution through microhomology-mediated end joining. Biochem. Biophys. Res. Commun. 502, 116–122 (2018).
Song, C. Q. et al. Adenine base editing in an adult mouse model of tyrosinaemia. Nat. Biomed. Eng. 4, 125–130 (2020).
Nishimasu, H. et al. Engineered CRISPR–Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).
Kim, N. et al. Prediction of the sequence-specific cleavage activity of Cas9 variants. Nat. Biotechnol. 38, 1328–1336 (2020).
Kim, H. K. et al. High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells. Nat. Biomed. Eng. 4, 111–124 (2020).
Kim, H. K. et al. SpCas9 activity prediction by DeepSpCas9, a deep learning-based model with high generalization performance. Sci. Adv. 5, eaax9249 (2019).
Wilkinson, P. D. et al. The polyploid state restricts hepatocyte proliferation and liver regeneration in mice. Hepatology 69, 1242–1258 (2019).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
Kim, D. Y., Moon, S. B., Ko, J. H., Kim, Y. S. & Kim, D. Unbiased investigation of specificities of prime editing systems in human cells. Nucleic Acids Res. 48, 10576–10589 (2020).
Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).
Gao, P. et al. Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression. Genome Biol. 22, 83 (2021).
Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-00901-y (2021).
DiCarlo, J. E., Mahajan, V. B. & Tsang, S. H. Gene therapy and genome surgery in the retina. J. Clin. Invest. 128, 2177–2188 (2018).
Sun, L., Li, J. & Xiao, X. Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Nat. Med. 6, 599–602 (2000).
Lai, Y. et al. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat. Biotechnol. 23, 1435–1439 (2005).
Ryu, S. M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2018).
Jo, D. H. et al. CRISPR–Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis. Sci. Adv. 5, eaax1210 (2019).
Cideciyan, A. V. Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy. Prog. Retin. Eye Res. 29, 398–427 (2010).
Sahel, J. A. Spotlight on childhood blindness. J. Clin. Invest. 121, 2145–2149 (2011).
Redmond, T. M. et al. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat. Genet. 20, 344–351 (1998).
Russell, S. et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390, 849–860 (2017).
Bainbridge, J. W. B. et al. Long-term effect of gene therapy on Leber’s congenital amaurosis. N. Engl. J. Med. 372, 1887–1897 (2015).
Jacobson, S. G. et al. Improvement and decline in vision with gene therapy in childhood blindness. N. Engl. J. Med. 372, 1920–1926 (2015).
Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019).
Cideciyan, A. V. et al. Effect of an intravitreal antisense oligonucleotide on vision in Leber congenital amaurosis due to a photoreceptor cilium defect. Nat. Med 25, 225–228 (2019).
Suh, S. et al. Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing. Nat. Biomed. Eng. 5, 169–178 (2021).
Jo, D. H. et al. Therapeutic adenine base editing corrects nonsense mutation and improves visual function in a mouse model of Leber congenital amaurosis. Preprint at bioRxiv https://doi.org/10.1101/2021.01.07.425822 (2021).
Pang, J. J. et al. Retinal degeneration 12 (rd12): a new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA). Mol. Vis. 11, 152–162 (2005).
Kweon, J. et al. Engineered prime editors with PAM flexibility. Mol. Ther. 29, 2001–2007 (2021).
Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97–110 (2020).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021).
Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 18, 358–378 (2019).
Li, C. & Samulski, R. J. Engineering adeno-associated virus vectors for gene therapy. Nat. Rev. Genet. 21, 255–272 (2020).
Song, M. et al. Sequence-specific prediction of the efficiencies of adenine and cytosine base editors. Nat. Biotechnol. 38, 1037–1043 (2020).
Du, D. et al. Genetic interaction mapping in mammalian cells using CRISPR interference. Nat. Methods 14, 577–580 (2017).
Shalem, O. et al. Genome-scale CRISPR–Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Kim, H. K. et al. In vivo high-throughput profiling of CRISPR–Cpf1 activity. Nat. Methods 14, 153–159 (2017).
Park, J., Lim, K., Kim, J. S. & Bae, S. Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33, 286–288 (2017).
Ramakrishna, S. et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat. Commun. 5, 3378 (2014).
Deverman, B. E. et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat. Biotechnol. 34, 204–209 (2016).
Park, S. W., Kim, J. H., Park, W. J. & Kim, J. H. Limbal approach-subretinal injection of viral vectors for gene therapy in mice retinal pigment epithelium. J. Vis. Exp. 102, 53030 (2015).
Prusky, G. T., Alam, N. M., Beekman, S. & Douglas, R. M. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest Ophthalmol. Vis. Sci. 45, 4611–4616 (2004).
Douglas, R. M. et al. Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system. Vis. Neurosci. 22, 677–684 (2005).
Acknowledgements
We thank S. Kwon and J. Park for helping with computational analyses; S. Park and Y. Kim for assisting with the experiments. This work was supported by the New Faculty Startup Fund from Seoul National University (D.H.J.); the Bio and Medical Technology Development Program of the National Research Foundation funded by the Korean government, Ministry of Science, ICT and Future Planning (NRF-2017M3A9B4062401 (H.H.K. and J.H.K.)); Brain Korea 21 Four Project for Medical Sciences (Yonsei University College of Medicine); the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2017R1A6A3A04004741 (D.H.J.)); the Medical Research Center from the National Research Foundation of Korea (2018R1A5A2025079 (H.H.K.)); grants from the National Research Foundation of Korea (2017R1A2B3004198 (H.H.K.) and 2020R1C1C1003284 (H.H.K.)); the Creative Materials Discovery Program through the National Research Foundation of Korea (NRF-2018M3D1A1058826 (J.H.K.)); the Korea Research Institute of Bioscience and Biotechnology(KRIBB) Research Initiative Program (KGM5362111 (J.H.K)); and the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (grant HI17C0676 (H.H.K.)).
Author information
Authors and Affiliations
Contributions
H.H.K. and J.H.K. conceived and designed the study; H.J. and J.H.S. evaluated prime-editing efficiencies and analysed DNA from mice under the supervision of H.H.K.; D.H.J. and C.S.C. conducted the eye-related animal experiments under the supervision of J.H.K; H.J., J. H. Shin, G.Y. and R.G. performed the high-throughput evaluation of PE2 efficiencies; J. H. Seo and S.-R.C. conducted immunofluorescence staining and histologic evaluations related to experiments associated with Fahmut/mut mice; D.K. performed the Digenome-seq and nDigenome-seq experiments; H.H.K., D.H.J., H.J. and J.H.K. wrote the manuscript with input from all authors.
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Yonsei University has filed a patent application based on this work and H.J. and H.H.K. are listed as inventors.
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Extended data
Extended Data Fig. 1 Schematic representation of the high-throughput evaluation of pegRNA activities.
A lentiviral plasmid library was prepared from a pool of oligonucleotides that contained pairs of pegRNA-encoding sequences and corresponding target sequences. Next, HEK293T cells were transduced with lentivirus generated from the plasmid library to construct a cell library and untransduced cells were removed by puromycin selection. This cell library was then transfected with a plasmid encoding NG-PE2, and untransfected cells were removed by blasticidin selection. Five days after the transfection, genomic DNA was isolated from the cells, PCR-amplified, and subjected to deep sequencing to determine prime editing efficiencies.
Extended Data Fig. 2 Prime editor 2 corrects the disease mutation and phenotype in Fahmut/mut mice.
a, Maps of vectors encoding PE2 components and a schematic representation of the experiments. Fahmut/mut mice underwent injection of plasmids encoding prime editor 2 components (that is, prime editor 2 and pegRNA) and were kept on water containing NTBC for 7 days. The day on which NTBC was initially withdrawn is defined as day 0. The mice were again provided with NTBC for five days, from day 7 to day 12, after the initial withdrawal of NTBC at day 0. At 60 days, the PE2-treated mice were euthanized and analyzed. Abbreviations in the vector maps are defined in the Supplementary Figure 2 legend. b, Body weight of Fahmut/mut mice injected with PE2 or phosphate-buffered saline (Saline, control). Body weights were normalized to the pre-injection weight. The number of mice n = 5 for the PE2 group and n = 3 for the saline group. Data are mean ± s.e.m. c, The level of wild-type Fah mRNA in the liver measured by quantitative RT-PCR using primers that hybridize to exons 8 and 9. WT, wild-type mice; Mut, Fahmut/mut mice; PE2, Fahmut/mut mice injected with plasmids encoding PE2 components. The number of mice n = 3 (WT), 3 (Mut), and 5 (PE2). **P = 0.0032. d, H&E staining (upper panels) and immunofluorescent staining for FAH protein (lower panels) in liver sections. Scale bars, upper panels, 100 μm; lower panels, 200 μm.
Extended Data Fig. 3 Correction of the LCA-causing mutation and phenotype using the most efficient pegRNA (id 157).
a, Frequencies of intended and unintended edits in the RPE of AAV-PE2-treated rd12 mice. The frequencies were normalized by subtracting the average frequency of such editing in the control group without PE2 treatment to exclude errors originating from PCR amplification and sequencing. Substitutions near the targeted nucleotide were evaluated over a 40-bp range centered on the targeted nucleotide. Indels were measured over a 60-bp range centered on the pegRNA nicking site. The red horizontal line represents the location where the normalized frequency = 0. Data are mean ± s.d. The number of mice n = 5. b, Substitution frequencies at each position of the target sequence, ranging from -20 bp to +20 bp from the targeted nucleotide, in the RPE of PE2-treated rd12 mice. The frequencies were normalized by subtracting the average edit frequencies in the RPE of rd12 mice without PE2 treatment to exclude errors originating from PCR amplification and sequencing. The red horizontal line represents the location where the normalized frequency = 0. Positions are numbered from the pegRNA nicking site. The targeted position is at +2. Data are mean ± s.d. The number of mice n = 5. c, Representative waveforms of dark-adapted ERG responses at 0 dB in wild-type (C57BL/6), uninjected control (rd12), and rd12 mice injected with PE2-expressing AAV (rd12-AAV-PE2). Scale bars, 30 ms (x-axis) and 50 μV (y-axis). d, Amplitudes of a-waves (left) and b-waves (right) of ERG responses of C57BL/6 and rd12 mice. Data are mean ± s.d. The number of mice n = 5. P-values from one-way ANOVA with post-hoc Tukey’s multiple comparison tests are shown. rd12-AAV-PE2, rd12 mice treated with AAV-PE2. e, Optomotor response test results. Data are mean ± s.d. The number of mice n = 5. P-values from one-way ANOVA with post-hoc Tukey’s multiple comparison tests are shown.
Supplementary information
Supplementary Information
Supplementary figures and table legends.
Supplementary Table 1
Predicted activities of SpCas9 and SpCas9-NG at nine target sequences for the prime editing of the tyrosinaemia-causing mutation.
Supplementary Table 2
Measured prime-editing efficiencies for the tested Fah pegRNAs, using a paired library approach.
Supplementary Table 3
Predicted activities of the sgRNA candidates for the PE3-directed correction of the tyrosinaemia-causing mutation.
Supplementary Table 4
Potential off-target sites and off-target effects evaluated in the study.
Supplementary Table 5
Predicted activities of the sgRNA candidates for the PE3-directed editing of Atp7b.
Supplementary Table 6
Prime-editing efficiencies of the Rpe65 pegRNAs, measured using a paired library approach.
Supplementary Table 7
Comparison of efficiencies and precision of genome-editing methods when genome-editing tools were delivered using hydrodynamic injections in a mouse model of hereditary tyrosinaemia.
Supplementary Table 8
Oligonucleotides used in the study.
Source data
Source Data Fig. 3
Unprocessed gels.
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Jang, H., Jo, D.H., Cho, C.S. et al. Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases. Nat Biomed Eng 6, 181–194 (2022). https://doi.org/10.1038/s41551-021-00788-9
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DOI: https://doi.org/10.1038/s41551-021-00788-9
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