Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos

Area of Science:

• Genetics
• Molecular Biology
• Reproductive Medicine

Background:

• Human germline gene correction offers potential to prevent genetic disease transmission.
• CRISPR-Cas9 gene editing in human embryos has raised concerns regarding mosaicism and loss of heterozygosity (LOH).
• LOH in edited embryos is hypothesized to stem from gene conversion or chromosomal abnormalities.

Purpose of the Study:

• To investigate the occurrence and mechanisms of LOH after targeted gene correction of a heterozygous mutation in the PLCZ1 gene in human embryos.
• To assess the integrity of the targeted chromosome following gene editing procedures.

Main Methods:

• Targeted CRISPR-Cas9 gene correction of a specific basepair substitution in the PLCZ1 gene in human embryos derived from mutant sperm.
• Genome-wide double-digest restriction site-associated DNA sequencing (ddRAD-seq) to assess chromosome integrity.
• Single-nucleotide polymorphism (SNP) assessment to detect short-range LOH events.

Main Results:

• In 36% of targeted embryos, only wild-type PLCZ1 alleles were detected, suggesting successful correction or off-target effects.
• Genome-wide analysis confirmed chromosomal integrity (no deletions >3 Mb or chromosome loss) in all analyzed embryos.
• Short-range LOH events (<10 Mb) were identified in two embryos via SNP analysis.

Conclusions:

• The findings support ongoing discussions about double-strand break repair mechanisms in early human embryos.
• Observed LOH events suggest the involvement of gene conversion or partial template-based homology-directed repair.
• Further research is needed to fully understand and mitigate LOH during human germline gene editing.