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FREQUENT GENE CONVERSION IN HUMAN EMBRYOS INDUCED BY DOUBLE STRAND BREAKS

Dan Liang, Nuria Marti Gutierrez, Tailai Chen, Yeonmi Lee, Sang-Wook Park, Hong Ma, Amy Koski, Riffat Ahmed, Hayley Darby, Ying Li, Crystal Van Dyken, Aleksei Mikhalchenko, Thanasup Gonmanee, Tomonari Hayama, Han Zhao, Keliang Wu, Jingye Zhang, Zhenzhen Hou, Jumi Park, Chong-Jai Kim, 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, Shoukhrat Mitalipov
doi: https://doi.org/10.1101/2020.06.19.162214
Dan Liang
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Nuria Marti Gutierrez
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Tailai Chen
2Center for Reproductive Medicine, Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, the Key laboratory of Reproductive Endocrinology, Ministry of Education, Jinan, Shandong 250001, China
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Yeonmi Lee
3Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea
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Sang-Wook Park
4Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon, 34047, Republic of Korea
5New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, 41061, Republic of Korea
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Hong Ma
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Amy Koski
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Riffat Ahmed
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Hayley Darby
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Ying Li
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Crystal Van Dyken
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Aleksei Mikhalchenko
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Thanasup Gonmanee
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Tomonari Hayama
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Han Zhao
2Center for Reproductive Medicine, Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, the Key laboratory of Reproductive Endocrinology, Ministry of Education, Jinan, Shandong 250001, China
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Keliang Wu
2Center for Reproductive Medicine, Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, the Key laboratory of Reproductive Endocrinology, Ministry of Education, Jinan, Shandong 250001, China
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Jingye Zhang
2Center for Reproductive Medicine, Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, the Key laboratory of Reproductive Endocrinology, Ministry of Education, Jinan, Shandong 250001, China
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Zhenzhen Hou
2Center for Reproductive Medicine, Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, the Key laboratory of Reproductive Endocrinology, Ministry of Education, Jinan, Shandong 250001, China
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Jumi Park
3Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea
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Chong-Jai Kim
3Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea
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Jianhui Gong
6Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China
7China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China
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Yilin Yuan
6Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China
7China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China
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Ying Gu
6Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China
7China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China
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Yue Shen
6Guangdong Provincial Key Laboratory of Genome Read and Write, BGI-Shenzhen, 518083, China
7China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China
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Susan B. Olson
8Department of Molecular and Medical Genetics and Knight Diagnostic Laboratories, Oregon Health & Science University, Portland, Oregon 97239, USA
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Hui Yang
9Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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David Battaglia
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Thomas O’Leary
10Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Sacha A. Krieg
10Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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David M. Lee
10Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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Diana H. Wu
10Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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P. Barton Duell
11Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, Oregon 97239, USA
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Sanjiv Kaul
11Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, Oregon 97239, USA
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Jin-Soo Kim
4Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon, 34047, Republic of Korea
12Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Republic of Korea
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Stephen B. Heitner
11Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, Oregon 97239, USA
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  • For correspondence: shoukhrat@gmail.com
Eunju Kang
3Stem Cell Center, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea
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  • For correspondence: shoukhrat@gmail.com
Zi-Jiang Chen
2Center for Reproductive Medicine, Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, the Key laboratory of Reproductive Endocrinology, Ministry of Education, Jinan, Shandong 250001, China
13Center for Reproductive Medicine, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200127, China
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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  • For correspondence: shoukhrat@gmail.com
Paula Amato
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
10Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
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  • For correspondence: shoukhrat@gmail.com
Shoukhrat Mitalipov
1Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA
2Center for Reproductive Medicine, Shandong University, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, the Key laboratory of Reproductive Endocrinology, Ministry of Education, Jinan, Shandong 250001, China
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  • For correspondence: shoukhrat@gmail.com
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Abstract

Applications of genome editing ultimately depend on DNA repair triggered by targeted double-strand breaks (DSBs). However, repair mechanisms in human cells remain poorly understood and vary across different cell types. Here we report that DSBs selectively induced on a mutant allele in heterozygous human embryos are repaired by gene conversion using an intact wildtype homolog as a template in up to 40% of targeted embryos. We also show that targeting of homozygous loci facilitates an interplay of non-homologous end joining (NHEJ) and gene conversion and results in embryos which carry identical indel mutations on both loci. Additionally, conversion tracks may expand bidirectionally well beyond the target region leading to an extensive loss of heterozygosity (LOH). Our study demonstrates that gene conversion and NHEJ are two major DNA DSB repair mechanisms in preimplantation human embryos. While gene conversion could be applicable for gene correction, extensive LOH presents a serious safety concern.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 20, 2020.
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FREQUENT GENE CONVERSION IN HUMAN EMBRYOS INDUCED BY DOUBLE STRAND BREAKS
Dan Liang, Nuria Marti Gutierrez, Tailai Chen, Yeonmi Lee, Sang-Wook Park, Hong Ma, Amy Koski, Riffat Ahmed, Hayley Darby, Ying Li, Crystal Van Dyken, Aleksei Mikhalchenko, Thanasup Gonmanee, Tomonari Hayama, Han Zhao, Keliang Wu, Jingye Zhang, Zhenzhen Hou, Jumi Park, Chong-Jai Kim, 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, Shoukhrat Mitalipov
bioRxiv 2020.06.19.162214; doi: https://doi.org/10.1101/2020.06.19.162214
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FREQUENT GENE CONVERSION IN HUMAN EMBRYOS INDUCED BY DOUBLE STRAND BREAKS
Dan Liang, Nuria Marti Gutierrez, Tailai Chen, Yeonmi Lee, Sang-Wook Park, Hong Ma, Amy Koski, Riffat Ahmed, Hayley Darby, Ying Li, Crystal Van Dyken, Aleksei Mikhalchenko, Thanasup Gonmanee, Tomonari Hayama, Han Zhao, Keliang Wu, Jingye Zhang, Zhenzhen Hou, Jumi Park, Chong-Jai Kim, 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, Shoukhrat Mitalipov
bioRxiv 2020.06.19.162214; doi: https://doi.org/10.1101/2020.06.19.162214

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