公开(公告)号：WO2013066438A3

公开(公告)日：2013-08-15

申请号：PCT/US2012047778

申请日：2012-07-22

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

Inventor： LIU DAVID R ,  GUILINGER JOHN PAUL ,  PATTANAYAK VIKRAM

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

CPC classification number: C12N9/22 ,  C12Q1/44 ,  C12Q1/6802 ,  C12Q1/6874 ,  C12Q2521/30

Abstract: 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 nulceases, 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. Analysis of the identified sites in cultured human cells revealed CCR5-224-induced mutagenesis at nine off-target loci. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. 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.

Abstract translation: 工程化核酸酶（例如，锌指核酸酶（ZFN），转录激活子样效应核酸酶（TALEN）等）是用于基因组操作和确定这些酶的脱靶切割位点的有希望的工具是非常有意义的。 我们开发了一种体外选择方法，其询问1011个DNA序列的活性二聚核酸酶例如ZFN和TALEN切割的能力。 该方法显示了数十万个DNA序列，一些存在于人类基因组中，可以通过分别靶向内源性人CCR5和VEGF-A基因的两个ZFNs CCR5-224和VF2468在体外切割。 在培养的人细胞中鉴定位点的分析显示在九个离靶位点上的CCR5-224诱导诱变。 类似地，我们观察到在培养的人细胞中VF2468切割的31个离靶位点。 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可以实现与ZFN中观察到的切割特异性相似或更高的裂解特异性。

公开(公告)号：WO2013066438A2

公开(公告)日：2013-05-10

申请号：PCT/US2012/047778

申请日：2012-07-22

Applicant: PRESIDENT AND FELLOWS OF HARVARD COLLEGE ,  LIU, David, R. ,  GUILINGER, John, Paul ,  PATTANAYAK, Vikram

Inventor： LIU, David, R. ,  GUILINGER, John, Paul ,  PATTANAYAK, Vikram

CPC classification number: C12N9/22 ,  C12Q1/44 ,  C12Q1/68 ,  C12Q1/6874 ,  C12Q1/6802 ,  C12Q2521/30

Abstract: 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 10 11 DNA sequences for their ability to be cleaved by active, dimeric nulceases, 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. Analysis of the identified sites in cultured human cells revealed CCR5-224-induced mutagenesis at nine off-target loci. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. 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.

Abstract translation: 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 非常感兴趣。 我们开发了一种体外选择方法，用于询问10 11 DNA序列被活性二聚体蛋白酶（例如ZFN和TALEN）切割的能力。 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，可以被两个靶向内源性人CCR5和VEGF-A的ZFNs，CCR5-224和VF2468在体外切割 基因，分别。 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可以实现与在ZFN中观察到的相似或更高的切割特异性。