Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
Nat Biotechnol. 2011 Aug 7;29(9):816-23. doi: 10.1038/nbt.1948.
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.
锌指核酸酶 (ZFNs) 通过在特定基因组位置诱导靶向 DNA 双链断裂 (DSB)，从而实现活细胞中的基因编辑。然而，用于描述 ZFNs 的全基因组特异性的策略仍然有限。我们表明，非同源末端连接可在 DSB 处捕获整合酶缺陷型慢病毒载体，从而标记这些瞬时事件。全基因组整合位点分析将靶向 CCR5 或 IL2RG 的四个 ZFN 对的实际体内切割活性进行了定位。通过深度测序对具有可重复检测到的核酸酶活性的基因座进行排名，使我们能够在这些位置监测体内 ZFN 特异性的程度。切割需要 ZFN 在与目标位点具有高度同源性的 DNA 上以特定的空间排列结合，并且仅容忍 ZFN 结合位点的个别位置的错配。尽管体内获得的共识结合序列与生化实验中获得的序列非常匹配，但体内切割位点的排名不能通过计算机预测。全面绘制体内 ZFN 活性图谱将有助于这些试剂在转化研究中的广泛应用。
