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bioRxiv · 10.1101/2022.10.29.514376

Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

Zou, R. S.·Liu, Y.·Reyes Gaido, O. E.·Konig, M. F.·Mog, B. J.·Shen, L. L.·Aviles-Vazquez, F.·Marin-Gonzalez, A.·Ha, T.
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Abstract

Discovery of off-target CRISPR-Cas genome editing activity in patient-derived cells and animal models is crucial for therapeutic applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) accumulates repair protein MRE11 at CRISPR-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation sequencing (ChIP-seq). This technique, termed DISCOVER-Seq+, discovered up to 5-fold more CRISPR off-target sites in immortalized cell lines, primary human cells, and mice compared to previous methods. We demonstrated applicability to ex vivo knock-in of a cancer-directed transgenic T-cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. DISCOVER-Seq+ is the most sensitive method to-date for discovering off-target genome editing in vivo.

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Zou, R. S., Liu, Y., Reyes Gaido, O. E., Konig, M. F., Mog, B. J., Shen, L. L., Aviles-Vazquez, F., Marin-Gonzalez, A., Ha, T.. 2022-10-30. Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+. https://doi.org/10.1101/2022.10.29.514376

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