RT Journal Article
SR Electronic
T1 Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.10.29.514376
DO 10.1101/2022.10.29.514376
A1 Roger S. Zou
A1 Yang Liu
A1 Oscar E. Reyes Gaido
A1 Maximilian F. Konig
A1 Brian J. Mog
A1 Leo L. Shen
A1 Franklin Aviles-Vazquez
A1 Alberto Marin-Gonzalez
A1 Taekjip Ha
YR 2022
UL http://biorxiv.org/content/early/2022/10/30/2022.10.29.514376.abstract
AB 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.Competing Interest StatementJohns Hopkins University has submitted a patent application on the method for off-target detection used in this study.