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Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence
Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241822/ https://www.ncbi.nlm.nih.gov/pubmed/28098181 http://dx.doi.org/10.1038/srep40638 |
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author | Zheng, Ting Hou, Yingzi Zhang, Pingjing Zhang, Zhenxi Xu, Ying Zhang, Letian Niu, Leilei Yang, Yi Liang, Da Yi, Fan Peng, Wei Feng, Wenjian Yang, Ying Chen, Jianxin Zhu, York Yuanyuan Zhang, Li-He Du, Quan |
author_facet | Zheng, Ting Hou, Yingzi Zhang, Pingjing Zhang, Zhenxi Xu, Ying Zhang, Letian Niu, Leilei Yang, Yi Liang, Da Yi, Fan Peng, Wei Feng, Wenjian Yang, Ying Chen, Jianxin Zhu, York Yuanyuan Zhang, Li-He Du, Quan |
author_sort | Zheng, Ting |
collection | PubMed |
description | Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA. |
format | Online Article Text |
id | pubmed-5241822 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-52418222017-01-23 Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence Zheng, Ting Hou, Yingzi Zhang, Pingjing Zhang, Zhenxi Xu, Ying Zhang, Letian Niu, Leilei Yang, Yi Liang, Da Yi, Fan Peng, Wei Feng, Wenjian Yang, Ying Chen, Jianxin Zhu, York Yuanyuan Zhang, Li-He Du, Quan Sci Rep Article Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA. Nature Publishing Group 2017-01-18 /pmc/articles/PMC5241822/ /pubmed/28098181 http://dx.doi.org/10.1038/srep40638 Text en Copyright © 2017, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Zheng, Ting Hou, Yingzi Zhang, Pingjing Zhang, Zhenxi Xu, Ying Zhang, Letian Niu, Leilei Yang, Yi Liang, Da Yi, Fan Peng, Wei Feng, Wenjian Yang, Ying Chen, Jianxin Zhu, York Yuanyuan Zhang, Li-He Du, Quan Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence |
title | Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence |
title_full | Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence |
title_fullStr | Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence |
title_full_unstemmed | Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence |
title_short | Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence |
title_sort | profiling single-guide rna specificity reveals a mismatch sensitive core sequence |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241822/ https://www.ncbi.nlm.nih.gov/pubmed/28098181 http://dx.doi.org/10.1038/srep40638 |
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