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Various repair events following CRISPR/Cas9-based mutational correction of an infertility-related mutation in mouse embryos

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Unpredictable genetic modifications and chromosomal aberrations following CRISPR/Cas9 administration hamper the efficacy of germline editing. Repair events triggered by double-strand DNA breaks (DSBs) besides non-homologous end joining and repair template-driven homology-directed repair have been insufficiently investigated in mouse. In this work, we are the first to investigate the precise repair mechanisms triggered by parental-specific DSB induction in mouse for paternal mutational correction in the context of an infertility-related mutation.

Methods

We aimed to correct a paternal 22-nucleotide deletion in Plcz1, associated with lack of fertilisation in vitro, by administrating CRISPR/Cas9 components during intracytoplasmic injection of Plcz1-null sperm in wild-type oocytes combined with assisted oocyte activation. Through targeted next-generation sequencing, 77 injected embryos and 26 blastomeres from seven injected embryos were investigated. In addition, low-pass whole genome sequencing was successfully performed on 17 injected embryo samples.

Results

Repair mechanisms induced by two different CRISPR/Cas9 guide RNA (gRNA) designs were investigated. In 13.73% (7/51; gRNA 1) and 19.05% (4/21; gRNA 2) of the targeted embryos, only the wild-type allele was observed, of which the majority (85.71%; 6/7) showed integrity of the targeted chromosome. Remarkably, for both designs, only in one of these embryos (1/7; gRNA 1 and 1/4; gRNA2) could repair template use be detected. This suggests that alternative repair events have occurred. Next, various genetic events within the same embryo were detected after single-cell analysis of four embryos.

Conclusion

Our results suggest the occurrence of mosaicism and complex repair events after CRISPR/Cas9 DSB induction where chromosomal integrity is predominantly contained.

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Data availability

Data is available upon reasonable request.

Code availability

Not applicable.

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Acknowledgements

The figures were created with BioRender.com. Graphs were created with GraphPad Prism.

Funding

We want to acknowledge the financial support by FWO-Vlaanderen (Flemish fund for scientific research) for B.B. (11C2821N), A.B. (1298722N), G.C. (11L8822N), B.M. (G077422N) and B.H. (G077422N).

Author information

Authors and Affiliations

  1. Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000, Ghent, Belgium

    B. Bekaert, A. Boel, A. Rybouchkin, G. Cosemans, S. Declercq, S. M. Chuva de Sousa Lopes, D. Stoop & B. Heindryckx

  2. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, 2333 ZA, the Netherlands

    S. M. Chuva de Sousa Lopes

  3. Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK

    J. Parrington

  4. Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Corneel Heymanslaan 10, 9000, Ghent, Belgium

    P. Coucke & B. Menten

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  1. B. Bekaert
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  2. A. Boel
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Contributions

B.B., A.B., B.M. and B.H. conceived and designed the project. B.B. performed most of the experiments, data analysis and wrote the manuscript. J.P. previously generated the mouse model and provided sperm samples. B.B., A.B., B.M. and P.C. designed and/or performed the genetic analyses. A.R. performed the CRISPR/Cas9 mouse germline injections. G.C. and S.D. helped with the experimental set-up. B.B., A.B., S.M.C.S.L, D.S., B.M., P.C. and B.H. interpreted the data. All authors reviewed the manuscript and approved the final version.

Corresponding author

Correspondence to B. Heindryckx.

Ethics declarations

All procedures involving live-animal handling and euthanasia of the animals were approved by the Ghent University Hospital Ethical Committee for Laboratory Animals (ECD 19-107 and ECD 19-107aanp) and were conform to all relevant regulatory standards.

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The authors declare no competing interests.

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Bekaert, B., Boel, A., Rybouchkin, A. et al. Various repair events following CRISPR/Cas9-based mutational correction of an infertility-related mutation in mouse embryos. J Assist Reprod Genet (2024). https://doi.org/10.1007/s10815-024-03095-9

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