Tsai Shengdar Q, Nguyen Nhu T, Malagon-Lopez Jose, Topkar Ved V, Aryee Martin J, Joung J Keith
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Nat Methods. 2017 Jun;14(6):607-614. doi: 10.1038/nmeth.4278. Epub 2017 May 1.
Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.
对脱靶效应进行灵敏检测对于将CRISPR-Cas9核酸酶转化为人类治疗方法而言至关重要。用于寻找脱靶位点的体外生化方法具有更高的可重复性和可扩展性等潜在优势，同时避免了与需要培养和操作活细胞的策略相关的局限性。在此，我们描述了用于体外切割效应测序报告的环化法（CIRCLE-seq），这是一种高度灵敏、测序高效的体外筛选策略，在全基因组范围内鉴定CRISPR-Cas9脱靶突变方面优于现有的基于细胞或生化的方法。与先前描述的体外方法不同，我们表明CIRCLE-seq可以使用广泛可用的新一代测序技术来实施，并且不需要参考基因组序列。重要的是，CIRCLE-seq可用于识别与细胞类型特异性单核苷酸多态性相关的脱靶突变，证明了生成个性化特异性图谱的可行性和重要性。CIRCLE-seq为鉴定CRISPR-Cas9全基因组脱靶突变提供了一种可及、快速且全面的方法。
