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Nature. 2018 Sep;561(7723):416-419. doi: 10.1038/s41586-018-0500-9. Epub 2018 Sep 12.

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

Author information

1
Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
2
Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA.
3
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
4
Department of Pathology, Harvard Medical School, Boston, MA, USA.
5
Advanced Medicines Safety, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
6
Quantitative Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
7
Wallenberg Laboratory and Sahlgrenska Center for Cardiovascular and Metabolic Research, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden.
8
San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.
9
Pathology Science, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
10
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
11
Advanced Medicines Safety, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
12
GE Healthcare Life Sciences, The Grove Centre, Amersham, UK.
13
Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden. marcello.maresca@astrazeneca.com.
14
Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
15
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
16
Department of Pathology, Harvard Medical School, Boston, MA, USA. jjoung@mgh.harvard.edu.

Abstract

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

PMID:
30209390
PMCID:
PMC6194229
DOI:
10.1038/s41586-018-0500-9
[Indexed for MEDLINE]
Free PMC Article

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