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Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos

Gregorio Alanis-Lobato, Jasmin Zohren, Afshan McCarthy, Norah M.E. Fogarty, Nada Kubikova, Emily Hardman, Maria Greco, Dagan Wells, James M.A. Turner, Kathy K. Niakan
doi: https://doi.org/10.1101/2020.06.05.135913
Gregorio Alanis-Lobato
aHuman Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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Jasmin Zohren
bSex Chromosome Biology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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Afshan McCarthy
aHuman Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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Norah M.E. Fogarty
aHuman Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
cCentre for Stem Cells and Regenerative Medicine, King’s College London, Guy’s Campus, Great Maze Pond, London SE1 9RT, UK
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Nada Kubikova
dUniversity of Oxford, Winchester House, Heatley Road, Oxford Science Park, Oxford OX4 4GE, UK
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Emily Hardman
aHuman Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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Maria Greco
eAncient Genomics Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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Dagan Wells
dUniversity of Oxford, Winchester House, Heatley Road, Oxford Science Park, Oxford OX4 4GE, UK
fJuno Genetics, Winchester House, Heatley Road, Oxford Science Park, Oxford OX4 4GE, UK
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James M.A. Turner
bSex Chromosome Biology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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Kathy K. Niakan
aHuman Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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  • For correspondence: kathy.niakan@crick.ac.uk
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Abstract

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germline. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and Cas9-only control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss-of-heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in approximately 22% of the human embryo cells analysed and spanned 4 to 20kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Competing Interest Statement

The authors have declared no competing interest.

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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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Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos
Gregorio Alanis-Lobato, Jasmin Zohren, Afshan McCarthy, Norah M.E. Fogarty, Nada Kubikova, Emily Hardman, Maria Greco, Dagan Wells, James M.A. Turner, Kathy K. Niakan
bioRxiv 2020.06.05.135913; doi: https://doi.org/10.1101/2020.06.05.135913
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Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos
Gregorio Alanis-Lobato, Jasmin Zohren, Afshan McCarthy, Norah M.E. Fogarty, Nada Kubikova, Emily Hardman, Maria Greco, Dagan Wells, James M.A. Turner, Kathy K. Niakan
bioRxiv 2020.06.05.135913; doi: https://doi.org/10.1101/2020.06.05.135913

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