清脆的
基因组编辑
生物
计算生物学
染色质
基因
染色质免疫沉淀
体内
HEK 293细胞
转基因
基因组
细胞生物学
分子生物学
遗传学
基因表达
发起人
作者
Roger S. Zou,Yang Liu,Oscar E. Reyes Gaido,Maximilian F. Konig,Brian J. Mog,Leo Shen,Franklin Aviles-Vazquez,Alberto Marín-González,Taekjip Ha
出处
期刊:Nature Methods
[Springer Nature]
日期:2023-04-06
卷期号:20 (5): 706-713
被引量:10
标识
DOI:10.1038/s41592-023-01840-z
摘要
Discovery of off-target CRISPR-Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR-Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate 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. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.
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