DNA Double Strand Breaks cause chromosome loss through sister chromatid tethering in human embryos

Abstract

Genome editing by DNA double-strand breaks (DSB) is currently being investigated as a tool to treat or even prevent heritable diseases. However, DNA repair mechanisms in the human embryo remain poorly understood and DSBs may result in chromosome loss. Here we provide evidence of whole and segmental chromosome loss in over one third of chromosomes 16, 17 and X targeted by CRISPR/Cas9-induced DNA DSB, including pericentromeric and mid-arm sites. Chromosomal changes were asymmetric relative to the Cas9 cut site: segmental losses occurred on both centric as well as acentric chromosome arms, while gains were exclusively found on acentric arms, suggesting that centromeres in broken chromosomes continued to mediate sister chromatid separation. Using this pattern of chromosomal errors, we were able to define new genomic coordinates of the active centromere on chromosome 16. Asymmetry was also found in the attrition of gDNA at the break site: attrition occurred centromeric of the DSB, while telomeric to the break, chromosomal ends were protected. Thus, spindle forces at centromeres and end tethering and protection at DSBs are antagonistic forces that interfere with accurate segregation of sister chromatids. Thereby, a single DSB is sufficient to result in the loss of a chromosome from the embryo. These results highlight the risks of aneuploidy in CRISPR/Cas9 genome editing, while also providing a mechanism for mitotically acquired aneuploidy caused by DNA breaks in human embryos.

DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility

“…A break site in the gene DPP10 shown in Fig. 2C , is in direct concordance with a break site found in human embryos ( 27 ). Importantly, and novel in this context, bovine fragile sites show delayed replication relative to random sites ( Fig.2F ).…”

DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility

“…A break site in the gene DPP10 shown in Fig. 2D, is in direct concordance with a break site found in human embryos 32 . Importantly, bovine fragile sites show delayed replication relative to random sites (Fig.…”