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Haplotype-phased common marmoset embryonic stem cells for genome editing using CRISPR/Cas9

View ORCID ProfileBo Zhou, Steve S. Ho, Louis C. Leung, Thomas R. Ward, Marcus Ho, Melanie J. Plastini, Scott C. Vermilyea, Marina E. Emborg, Thaddeus G. Golos, Megan A. Albertelli, Philippe Mourrain, Dimitri Perrin, Karen J. Parker, Alexander E. Urban
doi: https://doi.org/10.1101/373886
Bo Zhou
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
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  • ORCID record for Bo Zhou
Steve S. Ho
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
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Louis C. Leung
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
3Stanford Center for Sleep Sciences and Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
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Thomas R. Ward
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
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Marcus Ho
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
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Melanie J. Plastini
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
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Scott C. Vermilyea
4Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI 53715, USA
5Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA
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Marina E. Emborg
4Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI 53715, USA
5Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA
6Department of Medical Physics, University of Wisconsin-Madison, Madison, WI 53705, USA
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Thaddeus G. Golos
4Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI 53715, USA
7Departments of Comparative Biosciences and Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI 53715, USA
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Megan A. Albertelli
8Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA
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Philippe Mourrain
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
3Stanford Center for Sleep Sciences and Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
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Dimitri Perrin
9Science and Engineering Faculty, Queensland University of Technology, Brisbane, QLD 4001, Australia
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Karen J. Parker
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
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  • For correspondence: aeurban@stanford.edu kjparker@stanford.edu
Alexander E. Urban
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305, USA
2Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA
10Program on Genetics of Brain Function, Stanford Center for Genomics and Personalized Medicine, Tasha and John Morgridge Faculty Scholar, Stanford Child Health Research Institute, Stanford University, Stanford, CA, USA
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  • For correspondence: aeurban@stanford.edu kjparker@stanford.edu
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ABSTRACT

Due to anatomical and physiological similarities to humans, the common marmoset (Callithrix jacchus) is an ideal organism for the study human diseases. Researchers are currently leveraging genome-editing technologies such as CRISPR/Cas9 to genetically engineer marmosets for the in vivo biomedical modeling of human neuropsychiatric and neurodegenerative diseases. The genome characterization of these cell lines greatly reinforces these transgenic efforts. It also provides the genomic contexts required for the accurate interpretation of functional genomics data. We performed haplotype-resolved whole-genome characterization for marmoset ESC line cj367 from the Wisconsin National Primate Research Center. This is the first haplotype-resolved analysis of a marmoset genome and the first whole-genome characterization of any marmoset ESC line. We identified and phased single-nucleotide variants (SNVs) and Indels across the genome. By leveraging this haplotype information, we then compiled a list of cj367 ESC allele-specific CRISPR targeting sites. Furthermore, we demonstrated successful Cas9 Endonuclease Dead (dCas9) expression and targeted localization in cj367 as well as sustained pluripotency after dCas9 transfection by teratoma assay. Lastly, we show that these ESCs can be directly induced into functional neurons in a rapid, single-step process. Our study provides a valuable set of genomic resources for primate transgenics in this post-genome era.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • added co-author Megan Albertelli, DVM, PhD, DACLAM Associate Professor Department of Comparative Medicine Stanford University updated supp. table 3 (added phased genotype column)

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 October 21, 2020.
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Haplotype-phased common marmoset embryonic stem cells for genome editing using CRISPR/Cas9
Bo Zhou, Steve S. Ho, Louis C. Leung, Thomas R. Ward, Marcus Ho, Melanie J. Plastini, Scott C. Vermilyea, Marina E. Emborg, Thaddeus G. Golos, Megan A. Albertelli, Philippe Mourrain, Dimitri Perrin, Karen J. Parker, Alexander E. Urban
bioRxiv 373886; doi: https://doi.org/10.1101/373886
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Haplotype-phased common marmoset embryonic stem cells for genome editing using CRISPR/Cas9
Bo Zhou, Steve S. Ho, Louis C. Leung, Thomas R. Ward, Marcus Ho, Melanie J. Plastini, Scott C. Vermilyea, Marina E. Emborg, Thaddeus G. Golos, Megan A. Albertelli, Philippe Mourrain, Dimitri Perrin, Karen J. Parker, Alexander E. Urban
bioRxiv 373886; doi: https://doi.org/10.1101/373886

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