使用 CRISPR-Cas9 对低频和非重复染色体位点进行活细胞成像。

Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.

机构信息

Department of Physics, University of California at Berkeley, Berkeley, California 94720, USA.

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, USA.

出版信息

Nat Commun. 2017 Mar 14;8:14725. doi: 10.1038/ncomms14725.

Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells.

对染色质动力学进行成像对于理解基因组组织及其在转录调控中的作用至关重要。最近，CRISPR-Cas9 的 RNA 导向特性已被用于对活细胞内的染色质进行成像。然而，这些方法大多适用于高度重复的区域，而对低重复或无重复区域进行成像仍然是一个挑战。为了解决这个挑战，我们设计了整合多达 16 个 MS2 结合基序的单指导 RNA（sgRNA），以实现强大的荧光信号放大。这些工程化的 sgRNA 能够使用单个 sgRNA 对低重复含量的区域进行多色标记，以及使用多达四个独特的 sgRNA 对非重复区域进行标记。我们实现了在整个细胞周期中跟踪天然染色质位点，并确定转录活跃和不活跃区域在核内的差异定位。这些结果表明，我们的方法能够在活细胞中监测重复和非重复基因组区域的位置和动态。