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

Pinar Akcakaya, Maggie L. Bobbin, Jimmy A. Guo, Jose Malagon-Lopez, 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 () and J. Keith Joung ()
Additional contact information
Pinar Akcakaya: IMED Biotech Unit, AstraZeneca
Maggie L. Bobbin: Massachusetts General Hospital
Jimmy A. Guo: Massachusetts General Hospital
Jose Malagon-Lopez: Massachusetts General Hospital
Kendell Clement: Massachusetts General Hospital
Sara P. Garcia: Massachusetts General Hospital
Mick D. Fellows: IMED Biotech Unit, AstraZeneca
Michelle J. Porritt: IMED Biotech Unit, AstraZeneca
Mike A. Firth: IMED Biotech Unit
Alba Carreras: IMED Biotech Unit, AstraZeneca
Tania Baccega: IMED Biotech Unit, AstraZeneca
Frank Seeliger: Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca
Mikael Bjursell: IMED Biotech Unit, AstraZeneca
Shengdar Q. Tsai: Massachusetts General Hospital
Nhu T. Nguyen: Massachusetts General Hospital
Roberto Nitsch: Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca
Lorenz M. Mayr: IMED Biotech Unit, AstraZeneca
Luca Pinello: Massachusetts General Hospital
Mohammad Bohlooly-Y: IMED Biotech Unit, AstraZeneca
Martin J. Aryee: Massachusetts General Hospital
Marcello Maresca: IMED Biotech Unit, AstraZeneca
J. Keith Joung: Massachusetts General Hospital

Nature, 2018, vol. 561, issue 7723, 416-419

Abstract: 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.

Keywords: Gene-editing Nucleases; CRISPR-Cas9 Nuclease; Targeted Amplicon Sequencing; Supplementary Discussion; PCSK9 Gene (search for similar items in EconPapers)
Date: 2018
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Citations: View citations in EconPapers (2)

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DOI: 10.1038/s41586-018-0500-9

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