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Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments

Hera Canaj, Jeffrey A. Hussmann, Han Li, Kyle A. Beckman, Leeanne Goodrich, Nathan H. Cho, Yucheng J. Li, Daniel A. Santos, Aaron McGeever, Edna M. Stewart, Veronica Pessino, Mohammad A. Mandegar, Cindy Huang, Li Gan, Barbara Panning, Bo Huang, Jonathan S. Weissman, View ORCID ProfileManuel D. Leonetti
doi: https://doi.org/10.1101/841098
Hera Canaj
1Chan Zuckerberg Biohub, San Francisco, CA 94158
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Jeffrey A. Hussmann
2Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
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Han Li
2Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
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Kyle A. Beckman
1Chan Zuckerberg Biohub, San Francisco, CA 94158
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Leeanne Goodrich
3Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158
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Nathan H. Cho
1Chan Zuckerberg Biohub, San Francisco, CA 94158
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Yucheng J. Li
1Chan Zuckerberg Biohub, San Francisco, CA 94158
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Daniel A. Santos
2Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
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Aaron McGeever
1Chan Zuckerberg Biohub, San Francisco, CA 94158
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Edna M. Stewart
1Chan Zuckerberg Biohub, San Francisco, CA 94158
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Veronica Pessino
4Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158
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Mohammad A. Mandegar
5Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
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Cindy Huang
6Gladstone Institute of Neurological Disease, San Francisco, CA 94158
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Li Gan
6Gladstone Institute of Neurological Disease, San Francisco, CA 94158
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Barbara Panning
3Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158
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Bo Huang
1Chan Zuckerberg Biohub, San Francisco, CA 94158
4Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158
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Jonathan S. Weissman
2Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
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Manuel D. Leonetti
1Chan Zuckerberg Biohub, San Francisco, CA 94158
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  • ORCID record for Manuel D. Leonetti
  • For correspondence: jeffrey.hussmann@ucsf.edu manuel.leonetti@czbiohub.org
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Abstract

CRISPR/Cas technologies have transformed our ability to add functionality to the genome by knock-in of payload via homology-directed repair (HDR). However, a systematic and quantitative profiling of the knock-in integration landscape is still lacking. Here, we present a framework based on long-read sequencing and an integrated computational pipeline (knock-knock) to analyze knock-in repair outcomes across a wide range of experimental parameters. Our data uncover complex repair profiles, with perfect HDR often accounting for a minority of payload integration events, and reveal markedly distinct mis-integration patterns between cell-types or forms of HDR templates used. Our analysis demonstrates that the two sides of a given double-strand break can be repaired by separate pathways and identifies a major role for sequence micro-homology in driving donor mis-integration. Altogether, our comprehensive framework paves the way for investigating repair mechanisms, monitoring accuracy, and optimizing the precision of genome engineering.

Footnotes

  • https://github.com/jeffhussmann/knock-knock

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 November 13, 2019.
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Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments
Hera Canaj, Jeffrey A. Hussmann, Han Li, Kyle A. Beckman, Leeanne Goodrich, Nathan H. Cho, Yucheng J. Li, Daniel A. Santos, Aaron McGeever, Edna M. Stewart, Veronica Pessino, Mohammad A. Mandegar, Cindy Huang, Li Gan, Barbara Panning, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti
bioRxiv 841098; doi: https://doi.org/10.1101/841098
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Deep profiling reveals substantial heterogeneity of integration outcomes in CRISPR knock-in experiments
Hera Canaj, Jeffrey A. Hussmann, Han Li, Kyle A. Beckman, Leeanne Goodrich, Nathan H. Cho, Yucheng J. Li, Daniel A. Santos, Aaron McGeever, Edna M. Stewart, Veronica Pessino, Mohammad A. Mandegar, Cindy Huang, Li Gan, Barbara Panning, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti
bioRxiv 841098; doi: https://doi.org/10.1101/841098

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