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Limitations of gene editing assessments in human preimplantation embryos
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 coverag...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9992379/ https://www.ncbi.nlm.nih.gov/pubmed/36882397 http://dx.doi.org/10.1038/s41467-023-36820-6 |
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author | Liang, Dan Mikhalchenko, Aleksei Ma, Hong Marti Gutierrez, Nuria Chen, Tailai Lee, Yeonmi Park, Sang-Wook Tippner-Hedges, Rebecca Koski, Amy Darby, Hayley Li, Ying Van Dyken, Crystal Zhao, Han Wu, Keliang Zhang, Jingye Hou, Zhenzhen So, Seongjun Han, Jongsuk Park, Jumi Kim, Chong-Jai Zong, Kai Gong, Jianhui Yuan, Yilin Gu, Ying Shen, Yue Olson, Susan B. Yang, Hui Battaglia, David O’Leary, Thomas Krieg, Sacha A. Lee, David M. Wu, Diana H. Duell, P. Barton Kaul, Sanjiv Kim, Jin-Soo Heitner, Stephen B. Kang, Eunju Chen, Zi-Jiang Amato, Paula Mitalipov, Shoukhrat |
author_facet | Liang, Dan Mikhalchenko, Aleksei Ma, Hong Marti Gutierrez, Nuria Chen, Tailai Lee, Yeonmi Park, Sang-Wook Tippner-Hedges, Rebecca Koski, Amy Darby, Hayley Li, Ying Van Dyken, Crystal Zhao, Han Wu, Keliang Zhang, Jingye Hou, Zhenzhen So, Seongjun Han, Jongsuk Park, Jumi Kim, Chong-Jai Zong, Kai Gong, Jianhui Yuan, Yilin Gu, Ying Shen, Yue Olson, Susan B. Yang, Hui Battaglia, David O’Leary, Thomas Krieg, Sacha A. Lee, David M. Wu, Diana H. Duell, P. Barton Kaul, Sanjiv Kim, Jin-Soo Heitner, Stephen B. Kang, Eunju Chen, Zi-Jiang Amato, Paula Mitalipov, Shoukhrat |
author_sort | Liang, Dan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9992379 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-99923792023-03-09 Limitations of gene editing assessments in human preimplantation embryos Liang, Dan Mikhalchenko, Aleksei Ma, Hong Marti Gutierrez, Nuria Chen, Tailai Lee, Yeonmi Park, Sang-Wook Tippner-Hedges, Rebecca Koski, Amy Darby, Hayley Li, Ying Van Dyken, Crystal Zhao, Han Wu, Keliang Zhang, Jingye Hou, Zhenzhen So, Seongjun Han, Jongsuk Park, Jumi Kim, Chong-Jai Zong, Kai Gong, Jianhui Yuan, Yilin Gu, Ying Shen, Yue Olson, Susan B. Yang, Hui Battaglia, David O’Leary, Thomas Krieg, Sacha A. Lee, David M. Wu, Diana H. Duell, P. Barton Kaul, Sanjiv Kim, Jin-Soo Heitner, Stephen B. Kang, Eunju Chen, Zi-Jiang Amato, Paula Mitalipov, Shoukhrat Nat Commun Article 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. Nature Publishing Group UK 2023-03-07 /pmc/articles/PMC9992379/ /pubmed/36882397 http://dx.doi.org/10.1038/s41467-023-36820-6 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Liang, Dan Mikhalchenko, Aleksei Ma, Hong Marti Gutierrez, Nuria Chen, Tailai Lee, Yeonmi Park, Sang-Wook Tippner-Hedges, Rebecca Koski, Amy Darby, Hayley Li, Ying Van Dyken, Crystal Zhao, Han Wu, Keliang Zhang, Jingye Hou, Zhenzhen So, Seongjun Han, Jongsuk Park, Jumi Kim, Chong-Jai Zong, Kai Gong, Jianhui Yuan, Yilin Gu, Ying Shen, Yue Olson, Susan B. Yang, Hui Battaglia, David O’Leary, Thomas Krieg, Sacha A. Lee, David M. Wu, Diana H. Duell, P. Barton Kaul, Sanjiv Kim, Jin-Soo Heitner, Stephen B. Kang, Eunju Chen, Zi-Jiang Amato, Paula Mitalipov, Shoukhrat Limitations of gene editing assessments in human preimplantation embryos |
title | Limitations of gene editing assessments in human preimplantation embryos |
title_full | Limitations of gene editing assessments in human preimplantation embryos |
title_fullStr | Limitations of gene editing assessments in human preimplantation embryos |
title_full_unstemmed | Limitations of gene editing assessments in human preimplantation embryos |
title_short | Limitations of gene editing assessments in human preimplantation embryos |
title_sort | limitations of gene editing assessments in human preimplantation embryos |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9992379/ https://www.ncbi.nlm.nih.gov/pubmed/36882397 http://dx.doi.org/10.1038/s41467-023-36820-6 |
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