Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.
Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.
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DOI：
10.1093/nar/gkw066
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发表时间：
2016-05-19
影响因子：
14.9
通讯作者：
Sun Y
中科院分区：
文献类型：
--
作者：
Shao S;Zhang W;Hu H;Xue B;Qin J;Sun C;Sun Y;Wei W;Sun Y
染色体动力学的可视化对于理解许多基本的核内过程是重要的。有效和可靠的活细胞染色体位点的荧光标记可以实现这一目标。然而，目前的方法主要受到不足的标记通量，效率，灵活性以及光稳定性的限制。在这里，我们开发了一种基于CRISPR/Cas9系统的修饰的单向导RNA（sgRNA）实现双色染色体位点成像的新方法。通过对原始sgRNA进行结构引导工程改造来优化sgRNA的修饰，所述结构引导工程改造由结合荧光蛋白标记的效应物的RNA适体插入组成。通过标记和跟踪端粒，着丝粒和基因组位点，我们证明了新的方法很容易实现，并使强大的双色成像的基因组元素。重要的是，我们的数据还表明，RNA适体结合效应物的快速交换速率使得我们的基于sgRNA的标记方法比基于Cas9的标记方法更耐受光漂白。这对于持续、长期跟踪染色体动态至关重要。最后，由于我们的方法与其他活细胞基因组标记系统是互补的，因此可以将它们联合收割机组合成一个丰富的调色板，用于研究活细胞中的天然染色质组织和基因组超微结构动态。
Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.
DOI：
10.4161/nucl.28143
发表时间：
2014-01
期刊：
Nucleus (Austin, Tex.)
影响因子：
--
作者：
Pederson T
通讯作者：
Pederson T