In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.
In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.
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DOI：
10.1093/nar/gkt716
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发表时间：
2013-10
影响因子：
14.9
通讯作者：
Joung JK
中科院分区：
文献类型：
--
作者：
Sander JD;Ramirez CL;Linder SJ;Pattanayak V;Shoresh N;Ku M;Foden JA;Reyon D;Bernstein BE;Liu DR;Joung JK
基因编辑核酸酶能够靶向修饰活细胞中的DNA序列，从而促进内源基因座的有效敲除和精确编辑。工程化核酸酶也具有在脱靶作用位点引入突变的潜力。这种非预期的改变可能会混淆实验的解释，并可能对治疗应用的发展产生影响。最近，两种改进的方法来确定脱靶效应的锌指核酸酶（ZFN）的描述-一个使用在体外切割位点选择方法和其他利用插入的整合缺陷型慢病毒到核酸酶诱导的双链DNA断裂。然而，将这两种方法应用于靶向人CCR 5基因的ZFN对导致鉴定出很大程度上不重叠的脱靶位点，从而提高了可能存在额外脱靶位点的可能性。在这里，我们表明，从体外切割位点选择获得的ZFN切割谱的计算机提取可以极大地提高识别人类细胞中潜在脱靶位点的能力。我们改进的方法应该能够更全面地分析ZFN特异性。
Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.
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影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM
影响因子：
46.9
作者：
Gabriel, Richard;Lombardo, Angelo;von Kalle, Christof
通讯作者：
von Kalle, Christof
影响因子：
46.9
作者：
Miller, Jeffrey C.;Holmes, Michael C.;Rebar, Edward J.
通讯作者：
Rebar, Edward J.