An RNA-aptamer-based two-color CRISPR labeling system
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 r...
| Main Authors: | , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Springer Nature
2016
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| Online Access: | http://hdl.handle.net/1721.1/103876 https://orcid.org/0000-0003-2782-2509 |
| _version_ | 1826210312720744448 |
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| author | Wang, Siyuan Su, Jun-Han Zhang, Feng Zhuang, Xiaowei |
| author2 | Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences |
| author_facet | Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences Wang, Siyuan Su, Jun-Han Zhang, Feng Zhuang, Xiaowei |
| author_sort | Wang, Siyuan |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T14:47:50Z |
| format | Article |
| id | mit-1721.1/103876 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:47:50Z |
| publishDate | 2016 |
| publisher | Springer Nature |
| record_format | dspace |
| spelling | mit-1721.1/1038762022-09-29T10:37:58Z An RNA-aptamer-based two-color CRISPR labeling system Wang, Siyuan Su, Jun-Han Zhang, Feng Zhuang, Xiaowei Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences McGovern Institute for Brain Research at MIT Zhang, Feng 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. Jane Coffin Childs Memorial Fund for Medical Research (Fellowship) Howard Hughes Medical Institute (Investigator) 2016-08-10T14:52:59Z 2016-08-10T14:52:59Z 2016-05 2016-01 Article http://purl.org/eprint/type/JournalArticle 2045-2322 http://hdl.handle.net/1721.1/103876 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. https://orcid.org/0000-0003-2782-2509 en_US http://dx.doi.org/10.1038/srep26857 Scientific Reports Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/ application/pdf Springer Nature Scientific Reports |
| spellingShingle | Wang, Siyuan Su, Jun-Han Zhang, Feng Zhuang, Xiaowei An RNA-aptamer-based two-color CRISPR labeling system |
| title | An RNA-aptamer-based two-color CRISPR labeling system |
| title_full | An RNA-aptamer-based two-color CRISPR labeling system |
| title_fullStr | An RNA-aptamer-based two-color CRISPR labeling system |
| title_full_unstemmed | An RNA-aptamer-based two-color CRISPR labeling system |
| title_short | An RNA-aptamer-based two-color CRISPR labeling system |
| title_sort | rna aptamer based two color crispr labeling system |
| url | http://hdl.handle.net/1721.1/103876 https://orcid.org/0000-0003-2782-2509 |
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