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    EVALUATION AND IMPROVEMENT OF NUCLEASE CLEAVAGE SPECIFICITY
    1.
    发明公开
    EVALUATION AND IMPROVEMENT OF NUCLEASE CLEAVAGE SPECIFICITY 审中-公开
    评估和改进核化特性

    公开(公告)号：EP2734621A4

    公开(公告)日：2015-11-11

    申请号：EP12845790

    申请日：2012-07-22

    申请人： HARVARD COLLEGE ,  LIU DAVID R ,  GUILINGER JOHN PAUL ,  PATTANAYAK VIKRAM

    发明人： LIU DAVID R ,  GUILINGER JOHN PAUL ,  PATTANAYAK VIKRAM

    IPC分类号： C12N9/22 ,  A61K38/43 ,  C12N15/55 ,  C12Q1/44 ,  C12Q1/68 ,  C40B40/06

    CPC分类号： C12N9/22 ,  C12Q1/44 ,  C12Q1/6802 ,  C12Q1/6874 ,  C12Q2521/30

    摘要： Engineered nucleases (e.g., zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and others) are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for their ability to be cleaved by active, dimeric nucleases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future nuclease design. It was also observed that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.

    EVALUATION AND IMPROVEMENT OF NUCLEASE CLEAVAGE SPECIFICITY
    2.
    发明公开
    EVALUATION AND IMPROVEMENT OF NUCLEASE CLEAVAGE SPECIFICITY 审中-公开
    核酸酶切割特异性的评估和改进

    公开(公告)号：EP2734621A2

    公开(公告)日：2014-05-28

    申请号：EP12845790.0

    申请日：2012-07-22

    申请人： President and Fellows of Harvard College ,  Liu, David R. ,  Guilinger, John Paul ,  Pattanayak, Vikram

    发明人： LIU, David, R. ,  GUILINGER, John, Paul ,  PATTANAYAK, Vikram

    IPC分类号： C12N9/16 ,  C12N15/55 ,  C12Q1/44 ,  C12Q1/68 ,  C40B40/06 ,  A61K38/43

    CPC分类号： C12N9/22 ,  C12Q1/44 ,  C12Q1/68 ,  C12Q1/6874 ,  C12Q1/6802 ,  C12Q2521/30

    摘要： Engineered nucleases (e.g., zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and others) are promising tools for genome manipulation and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for their ability to be cleaved by active, dimeric nucleases, e.g., ZFNs and TALENs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs, CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGF-A genes, respectively. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future nuclease design. It was also observed that TALENs can achieve cleavage specificity similar to or higher than that observed in ZFNs.

    摘要翻译： 工程核酸酶（例如锌指核酸酶（ZFN），转录激活物样效应物核酸酶（TALEN）等）是用于基因组操作的有希望的工具并且确定这些酶的脱靶切割位点是非常有意义的。 我们开发了一种体外筛选方法，其询问10 11个DNA序列被活性二聚体抑制肽（例如ZFN和TALEN）裂解的能力。 该方法揭示了成千上万的DNA序列，一些存在于人类基因组中，可以通过两种分别靶向内源性人CCR5和VEGF-A基因的ZFNs，CCR5-224和VF2468在体外切割。 在培养的人类细胞中鉴定的位点的分析揭示了在9个脱靶位点处的CCR5-224诱导的诱变。 同样，我们在培养的人类细胞中观察到了VF2468切割的31个脱靶位点。 我们的研究结果建立了ZFN特异性的能量补偿模型，其中过量结合能量有助于脱靶ZFN切割并提出改进未来核酸酶设计的策略。 还观察到TALEN可以达到类似于或高于在ZFN中观察到的切割特异性。