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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2017
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.
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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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