锌指核酸酶（zinc-finger nucleases，ZFNs ）是一种能改变基因组特定位点的酶，这种改变基因组序列的技术也称为基因组编辑技术。如今，研究人员研究了基因组编辑技术的脱靶效应，新成果分别发表在8月在线出版的《自然—方法学》和《自然—生物技术》期刊上。
锌指核酸酶靶定在DNA上
借助内源DNA修复机制，锌指核酸酶可以精确改变高等动物的基因组，但精确性是这种技术或工具的核心。脱靶效应是指锌指核酸酶所改变的基因组位点不是最初设计的靶点，脱靶效应因此给这种技术或工具的应用带来问题，但科学家们一直没有在整个基因组中研究过这种问题。
两个独立研究小组分别描述了实施这种实验的方法，并报告了他们的发现。在《自然—方法学》期刊上，David Liu 和同事用一种试管内、高通量测序阵列研究了特定的两对锌指核酸酶的分裂。他们观察到了人类基因组中脱靶序列的分裂，看到当锌指核酸酶在细胞中表达时这些位点被分裂的证据。
在《自然—生物技术》期刊上，Luigi Naldini、 Christof von Kalle和同事报告，通过将一种细菌整合到基因组发生断裂的地方，他们鉴别出4对锌指核酸酶的断裂位点。虽然绝大多数切割位点是所期望的位点，但研究小组也发现了脱靶位点，同时也发现了未被计算机方法预测到的其他位点。
生物探索推荐英文摘要
Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection
Engineered zinc-finger nucleases (ZFNs) 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 cleavage by active, dimeric ZFNs. 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 VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224–induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. 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 ZFN design.
