核小体在位于核小体边缘的位点选择性抑制Cas9的脱靶活性。
Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge.
作者信息
Hinz John M, Laughery Marian F, Wyrick John J
机构信息
From the School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164.
From the School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
出版信息
J Biol Chem. 2016 Nov 25;291(48):24851-24856. doi: 10.1074/jbc.C116.758706. Epub 2016 Oct 18.
Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA substrates, but strongly inhibit cleavage of nucleosome substrates, particularly when the mismatch is in the sgRNA "seed" region. These findings indicate that nucleosomes may enhance Cas9 specificity by inhibiting cleavage of off-target sites at the nucleosome edge.
核小体影响Cas9在靶位点的结合和活性，但其在脱靶位点的影响尚不清楚。为了研究核小体如何在体外影响Cas9在脱靶位点的切割，我们使用了一种单导向RNA（sgRNA），该sgRNA先前已被证明能有效地引导Cas9在定位强烈的601核小体边缘进行切割。我们的数据表明，sgRNA与DNA靶标之间的单个错配对裸露DNA底物的Cas9切割影响相对较小，但强烈抑制核小体底物的切割，特别是当错配位于sgRNA的“种子”区域时。这些发现表明，核小体可能通过抑制核小体边缘脱靶位点的切割来增强Cas9的特异性。
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