FISHing Out the Details of CRISPR Genome Tracks.
FISHing Out the Details of CRISPR Genome Tracks.
复制标题
找出 CRISPR 基因组轨迹的详细信息。
DOI：
10.1016/j.bpj.2017.02.006
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发表时间：
2017
影响因子：
3.4
通讯作者：
Raj,Arjun
中科院分区：
文献类型：
--
作者：
Coté,AllisonJ;Raj,Arjun
基因组的动态空间组织在许多尺度上影响基因表达控制，从干细胞标记表达到影响单个基因的增强子-启动子对(1)。然而，单细胞缺乏动态测量，这使得确定基因组结构实际上是基因表达变化的原因，还是转录状态改变的影响具有挑战性。在这一期中，Guan等人(2)报告了一种方法，该方法通过允许研究人员开始实时观察活核中基因组的多个片段的运动，为实现这一技术目标迈出了第一步。CRISPR/CAS9的出现使得将荧光标记定位于活细胞中DNA的特定区域成为可能，但到目前为止，它的多重能力非常有限(3-5)。为了可视化几个基因组区域的相互作用，有必要同时成像这些DNA区域的运动。然而，同时对多个独特的较小区域进行成像仍然是一个挑战。CRISPR/Cas9 DNA成像通常基于Cas9-荧光蛋白融合，以DNA的特定单独区域为目标，基于作为序列特异性指南的共表达RNA的序列。然而，这种策略并不容易转化为区分多个遗传位点的能力：虽然通过表达许多不同的引导RNA来靶向DNA的许多区域是可能的，但不同的区域将无法相互区分，因为相同的荧光蛋白将被靶向所有这些区域。Guan等人(2)巧妙地克服了这一挑战，开发了一种方法，将活细胞中重复DNA的CRISPR/Cas9成像的动态信息与新的DNA FISH协议相结合，从而能够事后唯一地识别这些相同的基因座，从而极大地增加了活细胞CRISPR/Cas9基因座追踪的多重潜力(图1)。Guan等人(2)使用单色CRISPR/Cas9系统来针对多达四个独特的基因组基因座进行活细胞成像，并在固定后与迭代DNA FISH配对以识别哪些基因座是哪些。它们同时表达CRISPR和Cas9与荧光蛋白(EGFP)融合，以及许多不同的引导RNA，分别针对它们的四个座位。他们通过实时成像跟踪这些基因座，但在这一点上，无法确定每个斑点的身份。固定后，他们进行几轮DNA FISH，以追溯确定每个斑点的独特身份(图1)。DNA FISH可以识别固定细胞中的单个基因座，但受到可用于成像的荧光通道数量的限制。使用迭代DNA FISH，Guan等人(2)可以对一个基因座上的FISH进行成像，去除那些探针，然后进行另一次杂交，并以相同的荧光信号成像不同的基因座。传统上，这种方法受到DNA鱼类杂交时间较长的限制，但其速度的进步，从几天的杂交到几分钟，可能为大规模多重DNA鱼类打开大门，类似于近年来出现的许多使用迭代RNA FISH来描绘单细胞转录的技术(6-8)。使用单一颜色跟踪多个位点允许其他荧光成像方法报告可能是这些基因组运动的原因或结果的其他细胞状态，如基因表达变化或细胞周期状态。作为他们方法的原理证明应用，Guan等人(2)将他们的CRISPR/Cas9跟踪和DNA FISH技术与流行的细胞周期跟踪方法相结合，以检查单个基因组座位的复制时间。有了…，这种结合是不可能的
The dynamic spatial organization of the genome affects gene expression control at many scales, from stem cell marker expression to enhancer-promoter pairs affecting a single gene (1). The lack of dynamic measurements in single cells, however, has made it challenging to determine whether structure of the genome is actually a cause of these changes in gene expression, or an effect of altered transcriptional states. In this issue, Guan et al.(2) report a method that provides a first step toward this technical goal by allowing researchers to begin to look at the movement of multiple pieces of the genome in real time in living nuclei. The emergence of CRISPR/Cas9 has made it possible to target fluorescent labels to specific regions of DNA in live cells, but it has thus far had very limited multiplexing abilities (3–5). To visualize the interaction of several genomic regions, it will be necessary to simultaneously image the movement of those regions of DNA. Imaging multiple, unique smaller regions at once, however, remains a challenge. CRISPR/Cas9 DNA imaging is usually based on a Cas9-fluorescent protein fusion targeted toward a particular individual region of DNA based on the sequence of a coexpressed RNA that acts as a sequence-specific guide. However, this strategy does not easily translate to the ability to distinguish multiple genetic loci: while it is possible to target many regions of DNA by expressing many different guide RNAs, the different regions will be indistinguishable from one another because the same fluorescent protein will be targeted to all of them. Guan et al.(2) have cleverly surmounted this challenge by developing a method that combines the dynamic information from CRISPR/Cas9 imaging of repetitive DNA in live cells with new DNA FISH protocols that enable the unique identification of these same loci post facto, thus drastically increasing the multiplexing potential of live-cell CRISPR/Cas9 locus tracking (Fig. 1). Guan et al.(2) employ a single color CRISPR/Cas9 system to target up to four unique genomic loci for live cell imaging, and pair with iterative DNA FISH after fixation to identify which loci are which. They simultaneously express CRISPR and Cas9 fused with a fluorescent protein (EGFP), and many different guide RNAs that target each of their four loci. They track those loci via live imaging, but, at that point, cannot determine the identity of each spot. After fixation, they perform several rounds of DNA FISH to retroactively determine the unique identity of each spot (Fig. 1). DNA FISH can identify individual loci in fixed cells, but is limited to the number of fluorescent channels available for imaging. With iterative DNA FISH, Guan et al.(2) can image FISH on one locus, strip off those probes, and then perform another hybridization and image a different locus with the same fluorescent signature. Traditionally this approach was limited by long hybridization times for DNA FISH, but their advance in speed, from days of hybridization to minutes, may open the door for massively multiplexed DNA FISH, similar to many techniques that have arisen in recent years for profiling transcriptomes in single cells using iterative RNA FISH (6–8).The use of a single color to track multiple loci allows for other fluorescent imaging methods to report other cellular states that might be the cause or result of those genomic movements, such as gene expression changes or cell cycle state. As a proof-of-principle application of their method, Guan et al.(2) combine their CRISPR/Cas9 tracking and DNA FISH techniques with popular methods for cell cycle tracking to examine replication timing of individual genomic loci. This combination would have been impossible with …