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bioRxiv · 10.1101/469635

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Wienert, B.·Wyman, S. K.·Richardson, C. D.·Yeh, C. D.·Akcakaya, P.·Porritt, M. J.·Morlock, M.·Vu, J. T.·Kazane, K. R.·Watry, H. L.·Judge, L. M.·Conklin, B. R.·Maresca, M.·Corn, J. E.
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Abstract

Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

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Wienert, B., Wyman, S. K., Richardson, C. D., Yeh, C. D., Akcakaya, P., Porritt, M. J., Morlock, M., Vu, J. T., Kazane, K. R., Watry, H. L., Judge, L. M., Conklin, B. R., Maresca, M., Corn, J. E.. 2018-11-14. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. https://doi.org/10.1101/469635

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