Dynamics of CRISPR-Cas9 genome interrogation in living cells
Dynamics of CRISPR-Cas9 genome interrogation in living cells
复制标题
DOI：
10.1126/science.aac6572
复制
发表时间：
2015-11-13
期刊：
影响因子：
56.9
通讯作者：
Tjian, Robert
中科院分区：
文献类型：
--
作者：
Knight, Spencer C.;Xie, Liangqi;Tjian, Robert
RNA引导的CRISPR相关蛋白Cas9用于基因组编辑、转录调节和活细胞成像。Cas9-向导RNA复合物基于20个核苷酸的RNA-DNA互补性识别并切割双链DNA序列，但哺乳动物细胞中的靶标搜索机制尚不清楚。在这里，我们使用单粒子跟踪来可视化Cas9在活细胞中的扩散和染色质结合。我们表明，三维扩散主导了Cas9在体内的搜索，脱靶结合事件平均而言是短暂的（
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (