An RNA-aptamer-based two-color CRISPR labeling system
Name
An RNA-aptamer-based.pdf
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Author(s)
Wang, Siyuan
Su, Jun-Han
Zhang, Feng
Zhuang, Xiaowei
Date Issued
May 2016
Journal
Scientific Reports
Publisher
Springer Nature
Citation
Wang, Siyuan, Jun-Han Su, Feng Zhang, and Xiaowei Zhuang. "An RNA-aptamer-based two-color CRISPR labeling system." Scientific Reports 6, Article number: 26857 (2016), pp.1-6.
Version
Final published version
Abstract
The spatial organization and dynamics of chromatin play important roles in essential biological functions. However, direct visualization of endogenous genomic loci in living cells has proven to be laborious until the recent development of CRISPR-Cas9-based chromatin labeling methods. These methods rely on the recognition of specific DNA sequences by CRISPR single-guide RNAs (sgRNAs) and fluorescent–protein-fused catalytically inactive Cas9 to label specific chromatin loci in cells. Previously, multicolor chromatin labeling has been achieved using orthogonal Cas9 proteins from different bacterial species fused to different fluorescent proteins. Here we report the development of an alternative two-color CRISPR labeling method using only the well-characterized Streptococcus pyogenes Cas9, by incorporating MS2 or PP7 RNA aptamers into the sgRNA. The MS2 or PP7 aptamers then recruit the corresponding MS2 or PP7 coat proteins fused with different fluorescent proteins to the target genomic loci. Here we demonstrate specific and orthogonal two-color labeling of repetitive sequences in living human cells using this method. By attaching the MS2 or PP7 aptamers to different locations on the sgRNA, we found that extending the tetraloop and stem loop 2 of the sgRNA with MS2 or PP7 aptamers enhances the signal-to-background ratio of chromatin imaging.
MIT Department
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
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Creative Commons Attribution 4.0 International License
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DOI of Published Version
https://doi.org/10.1038/srep26857
