CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets.
CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets.
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DOI：
10.1038/nmeth.4278
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发表时间：
2017-06
期刊：
影响因子：
48
通讯作者：
Joung JK
中科院分区：
文献类型：
--
作者：
Tsai SQ;Nguyen NT;Malagon-Lopez J;Topkar VV;Aryee MJ;Joung JK
脱靶效应的灵敏检测对于将CRISPR-Cas9核酸酶转化为人类治疗剂是重要的。用于发现脱靶的体外生物化学方法提供了更大的可重复性和可扩展性的潜在优势，同时避免了与需要培养和操作活细胞的策略相关的限制。在这里，我们描述了CIRCLE-seq（通过测序体外报告C裂解效应的环化），这是一种高度灵敏、测序效率高的体外筛选策略，优于现有的基于细胞或生物化学的方法，用于鉴定CRISPR-Cas9全基因组脱靶突变。与先前描述的体外方法相比，我们表明CIRCLE-seq可以使用广泛使用的下一代测序技术来实践，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SNP相关的脱靶突变，证明了生成个性化特异性谱的可行性和重要性。CIRCLE-seq提供了迄今为止描述的用于鉴定CRISPR-Cas9的全基因组脱靶突变的最易获得、快速和全面的方法。
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 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 CIRCLE-seq (Circularization for In vitro Reporting of CLeavage Effects by sequencing), 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 sequence. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific SNPs, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides the most accessible, rapid and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9 described to date.
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影响因子：
48
作者：
Mali, Prashant;Esvelt, Kevin M.;Church, George M.
通讯作者：
Church, George M.
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
--
作者：
Brandsma I;Gent DC
通讯作者：
Gent DC
DOI：
10.1111/febs.13763
发表时间：
2016-09
期刊：
The FEBS journal
影响因子：
--
作者：
Lin J;Musunuru K
通讯作者：
Musunuru K
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin