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

Molecular Therapy, vol. 31, pp. 2326–2341

Abstract

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.

Authors 15

1. Bieke Bekaert

   Ghent University Hospital

   Affiliation as printed

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

2. Annekatrien Boel

   Ghent University Hospital

   Affiliation as printed

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

3. Lisa De Witte

   Ghent University

   Affiliation as printed

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

4. Winter Vandenberghe

   Ghent University Hospital

   Affiliation as printed

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

5. Mina Popovic

   Ghent University Hospital

   Affiliation as printed

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

6. Panagiotis Stamatiadis

   Ghent University Hospital

   Affiliation as printed

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

7. Gwenny Cosemans

   Ghent University Hospital

   Affiliation as printed

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

8. Lise Tordeurs

   Ghent University Hospital

   Affiliation as printed

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

9. A M De Loore

   Ghent University Hospital

   Affiliation as printed

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

10. Susana Marina Chuva de Sousa Lopes Aachen Department of Anatomy and Embryology

    Leiden University Medical Center · Ghent University Hospital

    Affiliation as printed

    Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium; Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands

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

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

11. Petra De Sutter

    Ghent University Hospital

    Affiliation as printed

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

12. Dominic Stoop

    Ghent University Hospital

    Affiliation as printed

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

13. Paul J. Coucke

    Ghent University

    Affiliation as printed

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

14. Björn Menten

    Ghent University

    Affiliation as printed

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

15. Björn Heindryckx corresponding

    Ghent University Hospital

    Affiliation as printed

    Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium. Electronic address: bjorn.heindryckx@ugent.be

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