Limitations of gene editing assessments in human preimplantation embryos

作者：Dan Liang, Aleksei E. Mikhalchenko, Hong Ma, Nuria Martí Gutiérrez, Tailai Chen, Yeonmi Lee, Sang-Wook Park, Rebecca Tippner-Hedges, Amy Koski, Hayley Darby, Ying Li, Crystal Van Dyken, Han Zhao, Keliang Wu, Jing‐ye Zhang, Zhenzhen Hou, Seongjun So, Jongsuk Han, Jumi Park, Chong-Jai Kim, Kai Zong, Jianhui Gong, Yilin Yuan, Ying Gu, Yue Shen, Susan B. Olson, Hui Yang, David E. Battaglia, Thomas O’Leary, Sacha A. Krieg, David M. Lee, Diana H. Wu, Paul Barton Duell, Sanjiv Kaul, Jin‐Soo Kim, Stephen B. Heitner, Eunju Kang, Zi‐Jiang Chen, Paula Amato, Shoukhrat M. Mitalipov · 发表于：Nature Communications · 年份：2023 · DOI：10.1038/s41467-023-36820-6 · 被引用次数：24 · 研究领域：CRISPR and Genetic Engineering、Animal Genetics and Reproduction、Pluripotent Stem Cells Research

Range of DNA repair in response to double-strand breaks 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 that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.