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Published on: April 2, 2020
Various repair events following CRISPR/Cas9-based mutational correction of an infertility-related mutation in mouse
B Bekaert1, A Boel1, A Rybouchkin1
1Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000, Ghent, Belgium.
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.
Insights
CRISPR/Cas9 gene editing in mice primarily results in complex DNA repair events and mosaicism, with chromosomal integrity largely maintained despite double-strand DNA breaks (DSBs). This study investigated these repair mechanisms for potential infertility mutation correction.
Area of Science:
- Molecular Biology
- Genetics
- Reproductive Biology
Background:
- CRISPR/Cas9 gene editing efficacy is limited by unpredictable genetic modifications and chromosomal aberrations.
- Repair mechanisms following double-strand DNA breaks (DSBs), including non-homologous end joining and homology-directed repair, are not fully understood in mouse germline editing.
- Investigating precise repair mechanisms is crucial for advancing germline editing technologies.
Purpose of the Study:
- To investigate precise DNA repair mechanisms following CRISPR/Cas9 induced DSBs in mouse embryos.
- To evaluate paternal mutational correction for an infertility-related mutation using CRISPR/Cas9.
- To understand the outcomes of DSB induction for germline editing applications.
Main Methods:
- Correction of a paternal 22-nucleotide deletion in the Plcz1 gene via CRISPR/Cas9 components during intracytoplasmic sperm injection.
- Administration of CRISPR/Cas9 components into Plcz1-null sperm injected into wild-type oocytes with assisted oocyte activation.
- Analysis of injected embryos and blastomeres using targeted next-generation sequencing and whole genome sequencing.
Main Results:
- The wild-type allele was observed in 13.73% (gRNA 1) and 19.05% (gRNA 2) of targeted embryos, with the majority showing chromosomal integrity.
- Repair template use was detected in only one embryo for each guide RNA design, suggesting alternative repair pathways.
- Single-cell analysis revealed diverse genetic events within individual embryos, indicating mosaicism.
Conclusions:
- CRISPR/Cas9 DSB induction in mouse embryos leads to complex repair events and mosaicism.
- Chromosomal integrity is predominantly maintained despite the induction of DSBs.
- Findings provide insights into the repair outcomes of CRISPR/Cas9-mediated germline editing.
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