Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
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DOI：
10.1126/science.aav9023
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发表时间：
2019-04-19
期刊：
影响因子：
56.9
通讯作者：
Corn, Jacob E.
中科院分区：
文献类型：
--
作者：
Wienert, Beeke;Wyman, Stacia K.;Corn, Jacob E.
CRISPR-Cas基因组编辑诱导靶向DNA损伤，但也可以影响非靶向位点。目前的脱靶发现方法使用纯化的DNA或特定的细胞模型，但无法在体内直接检测。我们开发了DISCOVER-Seq（原位Cas脱靶发现和测序验证），这是一种普遍适用的无偏脱靶鉴定方法，利用细胞和生物体中DNA修复因子的招募。追踪MRE11的精确募集揭示了单碱基分辨率下细胞中Cas活性的分子性质。DISCOVER-Seq与多种引导RNA格式和Cas酶类型一起工作，允许表征新的编辑工具。可以在细胞系和患者来源的诱导多能干细胞中以及在小鼠的腺病毒编辑过程中识别出脱靶，为在治疗性基因组编辑过程中在单个患者基因型中原位发现脱靶铺平了道路。
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.