Nucleosomes impede Cas9 access to DNA in vivo and in vitro
Nucleosomes impede Cas9 access to DNA in vivo and in vitro
复制标题
DOI：
10.7554/elife.12677
复制
发表时间：
2016-03-17
期刊：
影响因子：
7.7
通讯作者：
Weissman, Jonathan S.
中科院分区：
文献类型：
--
作者：
Horlbeck, Max A.;Witkowsky, Lea B.;Weissman, Jonathan S.
原核CRISPR（成簇规则间隔回文重复序列）相关蛋白Cas9已被广泛用作编辑、成像和调节真核基因组的工具。然而，我们对如何选择介导有效Cas9活性的单向导RNA（sgRNA）的理解是不完整的，因为我们缺乏对染色质如何影响Cas9靶向的了解。为了解决这一差距，我们分析了使用核酸酶活性或核酸酶死亡Cas9（dCas9）在人类细胞系中进行的大规模遗传筛选。我们观察到Cas9和dCas9的高活性sgRNA几乎仅在低核小体占有率的区域中发现。体外实验表明，核小体实际上直接阻碍Cas9结合和切割，而染色质重塑可以恢复Cas9接近。我们的研究结果揭示了真核染色质在决定这种移植细菌酶的靶向特异性方面的关键作用，并提供了选择Cas9靶位点的规则，这些靶位点与基于序列特性的靶位点不同并互补。
The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats) associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties.