Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos

Cell. 2020 Dec 10;183(6):1650-1664.e15. doi: 10.1016/j.cell.2020.10.025. Epub 2020 Oct 29.


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.

Keywords: Cas9; chromosome loss; double strand break repair; embryonic stem cells; germ-line gene editing; homologous recombination; human embryo; interhomolog recombination; microhomology mediated end joining; mitosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Animals
  • Base Sequence
  • Blastocyst / metabolism
  • CRISPR-Associated Protein 9 / metabolism*
  • Cell Cycle / genetics
  • Cell Line
  • Chromosome Deletion
  • Chromosomes, Human / genetics*
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair / genetics
  • Embryo Implantation / genetics
  • Embryo, Mammalian / metabolism*
  • Eye Proteins / genetics
  • Fertilization
  • Gene Editing
  • Gene Rearrangement / genetics
  • Genetic Loci
  • Genome, Human
  • Genotype
  • Heterozygote
  • Human Embryonic Stem Cells / metabolism
  • Humans
  • INDEL Mutation / genetics
  • Mice
  • Mitosis
  • Open Reading Frames / genetics
  • Polymorphism, Single Nucleotide / genetics


  • EYS protein, human
  • Eye Proteins
  • CRISPR-Associated Protein 9