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

• Published in: Molecular therapy Vol. 31; no. 8; pp. 2326 - 2341
• Main Authors: Bekaert, Bieke, Boel, Annekatrien, De Witte, Lisa, Vandenberghe, Winter, Popovic, Mina, Stamatiadis, Panagiotis, Cosemans, Gwenny, Tordeurs, Lise, De Loore, Athina-Maria, Chuva de Sousa Lopes, Susana Marina, De Sutter, Petra, Stoop, Dominic, Coucke, Paul, Menten, Björn, Heindryckx, Björn
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-08-2023; American Society of Gene & Cell Therapy
• Subjects: Alleles; chromosomal integrity; Chromosomes; CRISPR-Cas Systems; CRISPR-Cas9; gene conversion; Gene Editing - methods; germline genome editing; human embryo; Humans; Male; male infertility; mosaicism; Mutation; Original; Semen; chromosomal integrity; human embryo; CRISPR-Cas9; mosaicism; germline genome editing; gene conversion; male infertility
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1016/j.ymthe.2023.06.013

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targeted GENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.
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Heindryckx and colleagues describe that human germline mutational correction by CRISPR-Cas9 results in embryos showing additional mutagenesis as well as solely wild-type alleles. These embryos displayed integrity of the targeted chromosome, while short-range loss of heterozygosity could be detected, which could represent gene conversion events or partial template-based homology-directed repair.