An unbiased genome-wide analysis of zinc-finger nuclease specificity
An unbiased genome-wide analysis of zinc-finger nuclease specificity
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DOI：
10.1038/nbt.1948
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发表时间：
2011-09-01
影响因子：
46.9
通讯作者：
von Kalle, Christof
中科院分区：
文献类型：
--
作者：
Gabriel, Richard;Lombardo, Angelo;von Kalle, Christof
锌指核酸酶(ZFN)通过在特定的基因组位置诱导靶向DNA双链断裂(DSB)，在活细胞中实现基因编辑。然而，表征ZFN全基因组特异性的策略仍然有限。我们发现，非同源末端连接捕获整合酶缺陷的慢病毒载体在DSB，标记这些瞬时事件。全基因组整合位点分析描绘了针对CCR5或IL2RG的四个ZFN对的实际体内切割活性。通过深度测序对重复检测到的核酸酶活性的基因座进行排序，使我们能够监测这些位置上ZFN在体内的特异性程度。切割需要在DNA上特定的空间排列中结合ZFN，与预期的靶点高度同源，并且只允许ZFN结合位点的个别位置的错配。虽然在体内获得的共识结合序列与生化实验中获得的序列非常接近，但在Silico中无法预测体内切割位点的排序。体内ZFN活性的全面定位将有助于这些试剂在翻译研究中的广泛应用。
Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.