Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.
Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.
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DOI：
10.1371/journal.pone.0109213
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Pelletier J
中科院分区：
文献类型：
--
作者：
Cencic R;Miura H;Malina A;Robert F;Ethier S;Schmeing TM;Dostie J;Pelletier J
成簇规则间隔短回文重复序列（CRISPR）相关酶Cas9是一种RNA引导的核酸酶，已广泛适用于真核细胞中的基因组编辑。然而，人们对Cas9的体内靶向特异性知之甚少，大多数研究依赖于计算机模拟预测来定义潜在的脱靶编辑谱。使用染色质免疫沉淀然后测序（ChIP-seq），我们描绘了由靶向Trp 53基因座的两种不同的单向导（sg）RNA指导的催化失活的Cas9的全基因组结合全景。Cas9：sgRNA复合物能够加载到多个位点上，其中短种子区域邻近5′ NGG 3 ′原型间隔区邻近基序（PAM）。然而，在43个含有突变状态分析的种子区域的ChIP-seq位点中，我们发现仅在预期的靶位点和一个脱靶位点进行编辑。靶位点识别的体外分析显示，指导序列的5′端和PAM远端靶序列之间的相互作用对于有效地参与Cas9溶核活性是必要的，这解释了为什么脱靶编辑显著低于ChIP-seq数据的预期。
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.
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