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Application of prime editing to the correction of mutations and phenotypes in adult mice with liver and eye diseases

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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Fig. 1: A mouse model of tyrosinemia and high-throughput evaluation of pegRNAs.
Fig. 2: Evaluation of PE2 efficiencies using target sequence–containing cells.
Fig. 3: PE3 corrects the disease mutation and phenotype in Fahmut/mut mice.
Fig. 4: PE3 corrects the disease-causing mutation in a highly precise manner.
Fig. 5: AAV-mediated prime editing in the retina and RPE of wild-type mice.
Fig. 6: Identification of efficient pegRNAs for the correction of the LCA-causing mutation in rd12 mice, followed by subretinal injection of AAV-PE2 to correct the mutation in vivo.
Fig. 7: Subretinal injection of AAV-PE2 corrects the disease-causing mutation without any detectable off-target effects in rd12 mice.
Fig. 8: Restoration of RPE65 expression and improvement of visual function in rd12 mice after subretinal injection of AAVs encoding PE2 and the pegRNA (AAV-PE2).

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.

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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

Authors

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.

Corresponding authors

Correspondence to Jeong Hun Kim or Hyongbum Henry Kim.

Ethics declarations

Competing interests

Yonsei University has filed a patent application based on this work and H.J. and H.H.K. are listed as inventors.

Additional information

Peer review information Nature Biomedical Engineering thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Reporting Summary

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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