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In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations
Maggie L. Bobbin,
Jimmy A. Guo,
Jose M Lopez,
M. Kendell Clement,
Sara P. Garcia,
Mick D. Fellows,
Michelle J. Porritt,
Mike A. Firth,
Shengdar Q. Tsai,
Nhu T. Nguyen,
Lorenz M Mayr,
Martin J Aryee,
J. Keith Joung
Posted 27 Feb 2018
bioRxiv DOI: 10.1101/272724
Posted 27 Feb 2018
CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics but identifying unwanted off-target mutations remains an important requirement for clinical translation. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites. 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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