Limitations of gene editing assessments in human preimplantation embryos

Dan Liang, Aleksei Mikhalchenko, Hong Ma, Nuria Marti Gutierrez, Tailai Chen, Yeonmi Lee, Sang-Wook Park, Rebecca Tippner-Hedges, Amy Koski, Hayley Darby, Ying Li, Crystal Dyken, Han Zhao, Keliang Wu, Jingye 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 Battaglia, Thomas O’Leary, Sacha A. Krieg, David M. Lee, Diana H. Wu, P. Barton Duell, Sanjiv Kaul, Jin-Soo Kim, Stephen B. Heitner, Eunju Kang, Zi-Jiang Chen, Paula Amato and Shoukhrat Mitalipov ()
Additional contact information
Dan Liang: Oregon Health & Science University
Aleksei Mikhalchenko: Oregon Health & Science University
Hong Ma: Oregon Health & Science University
Nuria Marti Gutierrez: Oregon Health & Science University
Tailai Chen: Shandong University
Yeonmi Lee: CHA University
Sang-Wook Park: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu
Rebecca Tippner-Hedges: Oregon Health & Science University
Amy Koski: Oregon Health & Science University
Hayley Darby: Oregon Health & Science University
Ying Li: Oregon Health & Science University
Crystal Dyken: Oregon Health & Science University
Han Zhao: Shandong University
Keliang Wu: Shandong University
Jingye Zhang: Shandong University
Zhenzhen Hou: Shandong University
Seongjun So: CHA University
Jongsuk Han: CHA University
Jumi Park: University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu
Chong-Jai Kim: University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu
Kai Zong: Technology Center of Hefei Customs
Jianhui Gong: Guangdong Provincial Key Laboratory of Genome Read and Write
Yilin Yuan: Guangdong Provincial Key Laboratory of Genome Read and Write
Ying Gu: Guangdong Provincial Key Laboratory of Genome Read and Write
Yue Shen: Guangdong Provincial Key Laboratory of Genome Read and Write
Susan B. Olson: Oregon Health & Science University
Hui Yang: Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
David Battaglia: Oregon Health & Science University
Thomas O’Leary: Oregon Health & Science University, 3303 Southwest, Bond Avenue
Sacha A. Krieg: Oregon Health & Science University, 3303 Southwest, Bond Avenue
David M. Lee: Oregon Health & Science University, 3303 Southwest, Bond Avenue
Diana H. Wu: Oregon Health & Science University, 3303 Southwest, Bond Avenue
P. Barton Duell: Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road
Sanjiv Kaul: Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road
Jin-Soo Kim: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu
Stephen B. Heitner: Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road
Eunju Kang: CHA University
Zi-Jiang Chen: Oregon Health & Science University
Paula Amato: Oregon Health & Science University
Shoukhrat Mitalipov: Oregon Health & Science University

Nature Communications, 2023, vol. 14, issue 1, 1-15

Abstract: Abstract 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.

Date: 2023
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DOI: 10.1038/s41467-023-36820-6

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