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Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos
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Abstract
Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
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“and that microhomology-mediated end-joining (MMEJ) is the dominant repair pathway in this context (37).”
From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [37]
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“This suggests that MMEJ-derived large deletions (14, 37) are more likely than microhomology-mediated gene conversion in this context, though protein expression has yet to be fully characterised.”
From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [37]
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Metadata
- Authors
- Zuccaro, M. V., Xu, J., Mitchell, C., Marin, D., Zimmerman, R., Rana, B., Weinstein, E., King, R. T., Palmerola, K. L., Smith, M. E., Tsang, S. H., Goland, R., Jasin, M., Lobo, R., Treff, N., & Egli, D.
- Affiliations
- Columbia University Irving Medical Center (US)
- Genomic Prediction (United States) (US)
- Columbia University (US)
- Rutgers, The State University of New Jersey (US)
- Memorial Sloan Kettering Cancer Center (US)
- Corresponding author
- Egli, Dieter
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2020-12
- Volume
- 183
- Issue
- 6
- Pages
- 1650-1664.e15
- ISSN
- 0092-8674 (ISSN-L)
- OpenAlex
- W3095871885
- Cited by
- 303
- References
- 46
- Citations by year
- 2026: 27 · 2025: 30 · 2024: 51 · 2023: 64 · 2022: 63 · 2021: 57 · 2020: 11
- FWCI
- 15.99
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Cell (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
- PubMed (publishedVersion)
- Funders
- Irving Medical Center, Columbia University
- New York Stem Cell Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Keywords
- CRISPR/Cas9, human embryo, frameshift mutation, microhomology-mediated end joining, DNA double-strand break repair, zygote, paternal allele, chromosome loss, off-target cleavage, indels, embryo mosaicism, germline editing, inherited blindness
- MeSH terms
- Human Embryonic Stem Cells, Gene Editing, CRISPR-Associated Protein 9, Alleles, Animals, Base Sequence, Blastocyst, Cell Cycle, Cell Line, Chromosome Deletion, Chromosomes, Human, Embryo, Mammalian, Eye Proteins, Fertilization, Genotype, Heterozygote, Humans, Mitosis, Embryo Implantation, Gene Rearrangement, Genome, Human, Open Reading Frames, Polymorphism, Single Nucleotide, Mice, DNA Breaks, Double-Stranded, INDEL Mutation, Genetic Loci, DNA End-Joining Repair
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