Comparative Analysis of DNA Double Strand Break repair outcomes in Human Embryos and Embryonic Stem Cells

Range of DNA repair in response to double-strand breaks (DSBs) induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification (WGA) that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts (ADO) at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appeared as homozygous after WGA indicative of ADO. To overcome these limitations, we validated on-target modifications seen in gene edited human embryos in embryonic stem cells (ESCs). We show that, in addition to frequent indel mutations, biallelic DSBs can also produce large deletions at the target site. Moreover, some ESCs showed copy-neutral loss of heterozygosity (LOH) at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of LOH in ESCs was lower than in blastomeres, suggesting that ADO is a common WGA outcome limiting genotyping accuracy in human preimplantation embryos.