Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro
Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro
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DOI：
10.1021/acs.biochem.5b01108
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发表时间：
2015-12-08
期刊：
影响因子：
2.9
通讯作者：
Wyrick, John J.
中科院分区：
文献类型：
--
作者：
Hinz, John M.;Laughery, Marian F.;Wyrick, John J.
在真核细胞中进行 Cas9 基因组编辑期间，细菌 Cas9 酶会切割染色质内的 DNA 靶标。为了了解染色质如何影响 Cas9 靶向，我们在体外表征了 Cas9 对核小体底物的活性。我们发现，当 Cas9 核酸内切酶的靶位点位于核小体核心内时，其活性会受到强烈抑制。相反，核小体结构不会影响相邻接头 DNA 内靶位点的 Cas9 活性。对与核小体边缘部分重叠的靶位点的分析表明，原型间隔子相邻基序 (PAM) 的可及性是核小体上 Cas9 活性的关键决定因素。
During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.