Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos

Abstract

The correction of disease-causing mutations in human embryos could reduce the burden of inherited genetic disorders in the fetus and newborn, and improve the efficiency of fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here we evaluate the repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frame-shift 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. However, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, upon entry into mitosis, loss of one or both chromosomal arms. Thus, Cas9 allows for the modification of chromosomal content in human embryos in a targeted manner, which may be useful for the prevention of trisomies.

Simple and Efficient Custom Transcription Activator-Like Effector Gene Synthesis Via Twin Primer Assembly

“…The simplicity, versatility and easy accessibility of CRISPR/Cas9 make it popular in the world. However, several recent studies conducted in human embryos have raised safety concerns about heritable genetic changes [6,7]. These studies revealed frequent loss and reshuffling of the targeted chromosome and heightened concerns surrounding the uncertain risk of CRISPR/Cas9 editing.…”

“…These studies revealed frequent loss and reshuffling of the targeted chromosome and heightened concerns surrounding the uncertain risk of CRISPR/Cas9 editing. They also raise the possibility that the phenotype after gene editing is due to chromosome loss, rather than homologous recombination-based repair [6,7]. Thus several improvements should be made in the near future.…”

“…Thirdly, zinc finger nuclease and TALEN should also be considered since they are function as dimers and rely on protein-DNA recognition. Last but not least, the factors that decide chromosome end rejoining or loss and their mechanisms should be elucidated [6].…”

“…Therefore potential off-target cleavage activity still occurs both in vitro and in vivo [4,5]. Recently, Cas9-based gene editing in human embryos demonstrates frequent loss and reshuffling of the targeted chromosome [6,7]. In addition, Cas9 is much larger than the TALEN monomer, making it difficult to deliver into cells [8].…”

CRISPR/Cas9 from bench to bedside: what clinicians need to know before application?

“…Off-target effects are the most common challenge for all genome editing technologies, and CRISPR/Cas9 is no exception even for its crown of precision and efficiency. Only 22 days after the 2020 Noble Prize was announced, Dr. Dieter Egli's laboratory published an article entitled "Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos" in Cell, emphasizing the significant risk of aneuploidy and other adverse genetic consequences resulting from CRISPR/Cas9 gene editing in early human embryos [7]. This article demonstrated that approximately half of Cas9-induced double-strand breaks (DSB) remained unrepaired after manipulation, followed by chromosomal losses and hemizygous indels after mitosis due to off-target effects in both alleles.…”