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In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

Pinar Akcakaya, Maggie L. Bobbin, Jimmy A. Guo, Jose M. Lopez, M. Kendell Clement, Sara P. Garcia, Mick D. Fellows, Michelle J. Porritt, Mike A. Firth, Alba Carreras, Tania Baccega, Frank Seeliger, Mikael Bjursell, Shengdar Q. Tsai, Nhu T. Nguyen, Roberto Nitsch, Lorenz M. Mayr, Luca Pinello, Mohammad Bohlooly-Y, Martin J. Aryee, Marcello Maresca, J. Keith Joung
doi: https://doi.org/10.1101/272724
Pinar Akcakaya
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
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Maggie L. Bobbin
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
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Jimmy A. Guo
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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Jose M. Lopez
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
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M. Kendell Clement
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
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Sara P. Garcia
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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Mick D. Fellows
4New Drug Modalities, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom
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Michelle J. Porritt
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
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Mike A. Firth
5Discovery Biology, Quantitative Biology, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom
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Alba Carreras
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
6Current address: Wallenberg Laboratory and Sahlgrenska Center for Cardiovascular and Metabolic Research, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden
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Tania Baccega
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
7Current address: San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy
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Frank Seeliger
8Pathology Science, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
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Mikael Bjursell
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
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Shengdar Q. Tsai
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
9Current address: Department of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
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Nhu T. Nguyen
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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Roberto Nitsch
4New Drug Modalities, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Cambridge, United Kingdom
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Lorenz M. Mayr
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
10Current address: GE Healthcare Life Sciences, The Grove Centre, White Lion Road, Amersham, HP7 9LL, United Kingdom
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Luca Pinello
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
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Mohammad Bohlooly-Y
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
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Martin J. Aryee
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
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Marcello Maresca
1Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
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J. Keith Joung
2Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
3Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA
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Abstract

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

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Posted February 27, 2018.
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In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations
Pinar Akcakaya, Maggie L. Bobbin, Jimmy A. Guo, Jose M. Lopez, M. Kendell Clement, Sara P. Garcia, Mick D. Fellows, Michelle J. Porritt, Mike A. Firth, Alba Carreras, Tania Baccega, Frank Seeliger, Mikael Bjursell, Shengdar Q. Tsai, Nhu T. Nguyen, Roberto Nitsch, Lorenz M. Mayr, Luca Pinello, Mohammad Bohlooly-Y, Martin J. Aryee, Marcello Maresca, J. Keith Joung
bioRxiv 272724; doi: https://doi.org/10.1101/272724
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In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations
Pinar Akcakaya, Maggie L. Bobbin, Jimmy A. Guo, Jose M. Lopez, M. Kendell Clement, Sara P. Garcia, Mick D. Fellows, Michelle J. Porritt, Mike A. Firth, Alba Carreras, Tania Baccega, Frank Seeliger, Mikael Bjursell, Shengdar Q. Tsai, Nhu T. Nguyen, Roberto Nitsch, Lorenz M. Mayr, Luca Pinello, Mohammad Bohlooly-Y, Martin J. Aryee, Marcello Maresca, J. Keith Joung
bioRxiv 272724; doi: https://doi.org/10.1101/272724

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