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Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish

Lab Animal volume 54, pages 165–172 (2025)Cite this article

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Abstract

Zebrafish serve as a valuable model organism for studying human genetic diseases. While generating knockout lines is relatively straightforward, introducing precise disease-specific genetic variants by knock-in (KI) remains challenging. KI lines, however, enable more accurate studies of molecular and physiological consequences of genetic diseases. Their generation is often hampered by low editing efficiency (EE) and potential off-target effects. Here, we optimized conventional CRISPR–Cas9-mediated homology-directed repair (HDR) strategies for precise KI of genetic variants in zebrafish and compared their efficacy with prime editing, a recently developed technique that is not yet commonly used. Using next-generation sequencing, we determined KI EE by HDR for six unique base-pair substitutions in three different zebrafish genes. We assessed the effect of (1) varying Cas9 amounts, (2) HDR templates with chemical modifications to improve integration efficiency, (3) different microinjection procedures and (4) introduction of additional synonymous guide-blocking variants in the HDR template. Increasing Cas9 amounts augmented KI EE, with optimal injected amounts of Cas9 between 200 pg and 800 pg. The use of Alt-R HDR templates further increased KI EE, while guide-blocking modifications did not. Injecting components directly into the cell was not superior to injections into the yolk. Prime editing, however, increased EE up to fourfold and expanded the F0 founder pool for four targets compared with conventional HDR editing, with fewer off-target effects. Therefore, prime editing is a very promising methodology for improving the creation of precise genomic edits in zebrafish, facilitating the modeling of human diseases.

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Fig. 1: Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR components with 100, 200, 400 or 800 pg Cas9 into the yolk of one-cell-stage zebrafish embryos.
Fig. 2: Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR components into the cell or the yolk.
Fig. 3: Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR templates, Alt-R HDR templates with silent guide-blocking variants or unmodified HDR templates.
Fig. 4: Comparison of EE and IF obtained for each target by microinjection of Alt-R HDR components and PE components.
Fig. 5: F0 founder screen of 20 potential adult founder zebrafish generated by microinjection of HDR or PE components into the yolk of one-cell-stage zebrafish embryos.
Fig. 6: IF at top three predicted off-target locations.

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Data availability

All output values of BATCH-GE for IF and EE are provided and summarized in the supplementary tables. The raw fastq files that support the findings of this study are available from the corresponding authors upon request.

References

  1. Howe, K. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498–503 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Adhish, M. & Manjubala, I. Effectiveness of zebrafish models in understanding human diseases—a review of models. Heliyon 9, e14557 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Zang, L., Torraca, V., Shimada, Y. & Nishimura, N. Editorial: zebrafish models for human disease studies. Front. Cell Dev. Biol. 10, 861941 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  4. El-brolosy, M. A., Kontarakis, Z., Rossi, A. & Kuenne, C. Genetic compensation triggered by mutant mRNA degradation. Nature 568, 193–197 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Serobyan, V. et al. Transcriptional adaptation in Caenorhabditis elegans. eLife https://doi.org/10.7554/elife.50014 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Ma, Z. et al. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Nature 568, 259–263 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Pottie, L. et al. Loss of zebrafish atp6v1e1b, encoding a subunit of vacuolar ATPase, recapitulates human ARCL type 2C syndrome and identifies multiple pathobiological signatures. PLoS Genet. 17, e1009603 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yang, H. et al. Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks. Int. J. Mol. Sci. 21, 6461 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Salsman, J. & Dellaire, G. Precision genome editing in the CRISPR era. Biochem. Cell Biol. 95, 187–201 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Shakirova, A., Karpov, T., Komarova, Y. & Lepik, K. In search of an ideal template for therapeutic genome editing: a review of current developments for structure optimization. Front. Genome Ed. 5, 1068637 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Chai, R. et al. Gene editing by SSB/CRISPR–Cas9 ribonucleoprotein in bacteria. Int. J. Biol. Macromol. 278, 135065 (2024).

    Article  CAS  PubMed  Google Scholar 

  13. Bai, H. et al. CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish. BMC Genomics https://doi.org/10.1186/s12864-020-6493-4 (2020).

  14. Chen, F. et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat. Methods 8, 753–755 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Boel, A. et al. CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments. Dis. Models Mech. 11, dmm035352 (2018).

    Article  CAS  Google Scholar 

  16. Prill, K. & Dawson, J. F. Homology-directed repair in zebrafish: witchcraft and wizardry? Front. Mol. Biosci. 7, 595474 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Prykhozhij, S. V. & Berman, J. N. Mutation knock-in methods using single-stranded dna and gene editing tools in zebrafish. Methods Mol. Biol. 2707, 279–303 (2024).

    Article  CAS  PubMed  Google Scholar 

  18. Prykhozhij, S. V. et al. Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Nucleic Acids Res. 46, 17 (2018).

    Article  Google Scholar 

  19. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhao, Z., Shang, P., Mohanraju, P. & Geijsen, N. Prime editing: advances and therapeutic applications. Trends Biotechnol. 41, 1000–1012 (2023).

    Article  CAS  PubMed  Google Scholar 

  21. Gistelinck, C. et al. Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies. Proc. Natl Acad. Sci. USA 115, E8037–E8046 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Vierstraete, J. et al. Accurate quantification of homologous recombination in zebrafish: brca2 deficiency as a paradigm. Sci. Rep. 7, 16518 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Vierstraete, J., Fieuws, C., Willaert, A., Vral, A. & Claes, K. B. M. Zebrafish as an in vivo screening tool to establish PARP inhibitor efficacy. DNA Repair 97, 103023 (2021).

    Article  CAS  PubMed  Google Scholar 

  24. Van Damme, T. et al. Mutations in ATP6V1E1 or ATP6V1A cause autosomal-recessive cutis laxa. Am. J. Hum. Genet. 100, 216–227 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Richardson, M. E. et al. Strong functional data for pathogenicity or neutrality classify BRCA2 DNA-binding-domain variants of uncertain significance. Am. J. Hum. Genet. 108, 458–468 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chen, Q., Zhang, Y. & Yin, H. Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing. Adv. Drug Deliv. Rev. 168, 246–258 (2021).

    Article  CAS  PubMed  Google Scholar 

  27. Schubert, M. S. et al. Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair. Sci. Rep. 11, 19482 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Okamoto, S., Amaishi, Y., Maki, I., Enoki, T. & Mineno, J. Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Sci. Rep. 9, 4811 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liang, X., Potter, J., Kumar, S., Ravinder, N. & Chesnut, J. D. Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J. Biotechnol. 241, 136–146 (2017).

    Article  CAS  PubMed  Google Scholar 

  31. O'Brien, A. R., Wilson, L. O. W., Burgio, G. & Bauer, D. C. Unlocking HDR-mediated nucleotide editing by identifying high-efficiency target sites using machine learning. Sci. Rep. 9, 2788 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Wang, K. et al. Efficient generation of orthologous point mutations in pigs via CRISPR-assisted ssODN-mediated homology-directed repair. Mol. Ther. Nucleic Acids 5, e396 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mao, Z., Bozzella, M., Seluanov, A. & Gorbunova, V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair 7, 1765–1771 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ferree, P. L., Deneke, V. E. & Di Talia, S. Measuring time during early embryonic development. Semin. Cell Dev. Biol. 55, 80–88 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Aksoy, Y. A. et al. Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos. Commun. Biol. 2, 198 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Yuan, S. & Sun, Z. Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J. Vis. Exp. https://doi.org/10.3791/1113 (2009).

  37. Kroll, F. et al. A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes. eLife https://doi.org/10.7554/elife.59683 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Cartegni, L., Chew, S. L. & Krainer, A. R. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Cartegni, L., Wang, J., Zhu, Z., Zhang, M. Q. & Krainer, A. R. ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res. 31, 3568–3571 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Findlay, G. M. et al. Accurate classification of BRCA1 variants with saturation genome editing. Nature 562, 217–222 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Iwata, H. & Gotoh, O. Comparative analysis of information contents relevant to recognition of introns in many species. BMC Genomics 12, 45 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Jaganathan, K. et al. Predicting splicing from primary sequence with deep learning. Cell 176, 535–548 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Li, G. et al. Highly efficient and precise base editing in discarded human tripronuclear embryos. Protein Cell 8, 776–779 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR–Cas9 system. Nat. Commun. 8, 118 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ponnienselvan, K. et al. Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency. Nucleic Acids Res. 51, 6966–6980 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2022).

    Article  CAS  PubMed  Google Scholar 

  49. Zhang, W. et al. Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions. eLife 12, RP90948 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Mathis, N. et al. Predicting prime editing efficiency and product purity by deep learning. Nat. Biotechnol. 41, 1151–1159 (2023).

    Article  CAS  PubMed  Google Scholar 

  51. Tsai, H. H. et al. Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR–Cas9-mediated genome editing. Nat. Commun. 14, 5183 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Aleström, P. et al. Zebrafish: housing and husbandry recommendations. Lab Anim. 54, 213–224 (2020).

    Article  PubMed  Google Scholar 

  53. Westerfield, M. The Zebrafish Book; A Guide for the Laboratory Use of Zebrafish (Danio rerio) 5th edn (Univ. of Oregon Press, 2007).

  54. Chow, R. D., Chen, J. S., Shen, J. & Chen, S. A web tool for the design of prime-editing guide RNAs. Nat. Biomed. Eng. 5, 190–194 (2021).

    Article  CAS  PubMed  Google Scholar 

  55. De Leeneer, K. et al. Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice. Hum. Mutat. 36, 379–387 (2015).

    Article  PubMed  Google Scholar 

  56. Boel, A. et al. BATCH-GE: batch analysis of next-generation sequencing data for genome editing assessment. Sci. Rep. 6, 30330 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the Zebrafish Facility Ghent Core at Ghent University, and particularly K. Vermeulen for the diligent care for the zebrafish. We also thank the VIB Protein Core for the generation of the prime editor and J. K. Joung for the donation of the pET-PE2-His plasmid (Addgene plasmid no. IK1822). This research was funded by a grant from the Research Foundation – Flanders (grant no. FWOOPR2020009501) to B.C. and A.W., a Concerted Research Action grant from the Ghent University Special Research Fund (grant no. BOF GOA019-21) to B.C. and a Ghent University Special Research Fund (grant no. BOF21/DOC/242) to E.D.N. B.C. is a senior clinical investigator of the Research Foundation – Flanders.

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Author notes

  1. These authors contributed equally: Michiel Vanhooydonck, Elyne De Neef.

  2. These authors jointly supervised this work: Andy Willaert, Bert Callewaert, Kathleen B. M. Claes.

Authors and Affiliations

  1. Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium

    Michiel Vanhooydonck, Elyne De Neef, Hanna De Saffel, Andy Willaert, Bert Callewaert & Kathleen B. M. Claes

  2. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

    Michiel Vanhooydonck, Elyne De Neef, Hanna De Saffel, Andy Willaert, Bert Callewaert & Kathleen B. M. Claes

  3. Cancer Research Institute Ghent, Ghent, Belgium

    Elyne De Neef & Kathleen B. M. Claes

  4. Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University, Ghent, Belgium

    Annekatrien Boel

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  1. Michiel Vanhooydonck
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Contributions

Conceptualization: A.B., A.W., B.C. and K.B.M.C. Experimental procedures: M.V., E.D.N. and H.D.S. Data analysis: M.V., E.D.N. and H.D.S. Visualization and writing: M.V. and E.D.N. Original draft preparation: M.V. and E.D.N. Review and editing: M.V., E.D.N., A.W., B.C. and K.B.M.C. Supervision: A.B., A.W., B.C. and K.B.M.C. Funding acquisition: A.W., B.C. and K.B.M.C.

Corresponding authors

Correspondence to Bert Callewaert or Kathleen B. M. Claes.

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Supplementary Methods: generation of the PE protein.

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Vanhooydonck, M., De Neef, E., De Saffel, H. et al. Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish. Lab Anim 54, 165–172 (2025). https://doi.org/10.1038/s41684-025-01560-1

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