Off-Target Analysis of Zinc Finger Nucleases. Danio rerio

Zinc Finger Nucleases (ZFNs) facilitate precise editing of DNA enabling targeted genomic modifications in vivo. ZFNs have been employed to obtain genetically modified plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double stranded breaks at off-target sites within the genome. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites within the zebrafish genome using Illumina sequencing. Only a minority of these off-target sites displayed significant lesion frequency with kdrl ZFNs. Furthermore, we find that active off-target sites appear to be defined by the thermodynamics of zinc finger-DNA recognition. Surprisingly, we observed that the zinc finger protein specificity and the choice of the engineered dimerization domain of the FokI nuclease could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both ZFP-dependent and –independent mechanisms of potential improvement for engineering ZFNs with higher levels of precision. Overall design: Examined lesions at 141 off-target sites for various treatments of ZFNs and compare to the untreated sample stage 1: raw read but missing quality values stage 2: fastq files available from SRA

锌指核酸酶（Zinc Finger Nucleases, ZFNs）可介导精准的DNA编辑，实现体内靶向基因组修饰。ZFNs已被用于培育转基因动植物，基于细胞的ZFN疗法目前正处于临床试验阶段。然而，多数ZFN会呈现剂量依赖性毒性，推测其原因是在基因组内的脱靶位点产生了非预期的双链断裂。为评估影响ZFN功能特异性的参数，本研究对比了靶向斑马鱼kdrl基因座的ZFN变体的体内活性，这些变体同时具备高靶向活性与剂量依赖性毒性。我们通过Illumina测序技术，检测斑马鱼基因组内141个潜在脱靶位点的突变频率，以此评估这些ZFN的功能特异性。在这些潜在脱靶位点中，仅有少数在kdrl ZFN处理后呈现出显著的突变频率。此外，本研究发现，具有活性的脱靶位点似乎由锌指蛋白与DNA识别的热力学特性所决定。令人意外的是，本研究观察到，锌指蛋白（Zinc Finger Protein, ZFP）的特异性以及FokI核酸酶的工程化二聚化结构域选择，均可独立影响这些ZFN的保真度。本研究结果可为基因组内潜在脱靶位点的评估提供参考，并指出了两类可用于工程化改造高精准度ZFN的潜在优化机制：依赖锌指蛋白的机制与不依赖锌指蛋白的机制。整体实验设计：针对不同ZFN处理组，检测141个脱靶位点的突变情况，并与未处理样本进行对照。阶段1：仅包含原始测序读段，缺失质量值信息；阶段2：可从SRA获取FASTQ格式文件。