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

B Bekaert; A Boel; A Rybouchkin; G Cosemans; S Declercq; S M Chuva de Sousa Lopes; J Parrington; D Stoop; P Coucke; B Menten; B Heindryckx

doi:10.1007/s10815-024-03095-9

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

J Assist Reprod Genet. 2024 Apr 1. doi: 10.1007/s10815-024-03095-9. Online ahead of print.

Authors

B Bekaert¹, A Boel¹, A Rybouchkin¹, G Cosemans¹, S Declercq¹, S M Chuva de Sousa Lopes^{1

2}, J Parrington³, D Stoop¹, P Coucke⁴, B Menten⁴, B Heindryckx⁵

Affiliations

¹ Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000, Ghent, Belgium.
² Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, 2333 ZA, the Netherlands.
³ Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK.
⁴ Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Corneel Heymanslaan 10, 9000, Ghent, Belgium.
⁵ Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000, Ghent, Belgium. Bjorn.Heindryckx@UGent.be.

PMID: 38557805
DOI: 10.1007/s10815-024-03095-9

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.

Keywords: CRISPR/Cas9; Chromosomal integrity; Germline genome editing; Male infertility; Mosaicism; Mouse embryo.