CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment

Abstract The CRISPR/Cas9 complex, a bacterial immune response system, has been widely adopted for RNA-guided genome editing and transcription regulation in applications such as targeted genome modification and site-directed mutagenesis. However, the physical basis for its target specificity is not f...

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Autores principales:	Xiaojun Xu, Dongsheng Duan, Shi-Jie Chen
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2ff2b28d1acd498f8b5cb6119d43ad02
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spelling	oai:doaj.org-article:2ff2b28d1acd498f8b5cb6119d43ad022021-12-02T15:06:12ZCRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment10.1038/s41598-017-00180-12045-2322https://doaj.org/article/2ff2b28d1acd498f8b5cb6119d43ad022017-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-00180-1https://doaj.org/toc/2045-2322Abstract The CRISPR/Cas9 complex, a bacterial immune response system, has been widely adopted for RNA-guided genome editing and transcription regulation in applications such as targeted genome modification and site-directed mutagenesis. However, the physical basis for its target specificity is not fully understood. In this study, based on a statistical mechanical analysis for the whole ensemble of sgRNA-target complex conformations, we identify a strong correlation between Cas9 cleavage efficiency and the stability of the DNA-RNA (R-loop) complex structures, with a Pearson correlation coefficient ranging from 0.775 to 0.886 for the tested systems. The finding leads to quantitative insights into important experimental results, such as the greater Cas9 tolerance to single-base mismatches in PAM-distal region than to PAM-proximal region and the high specificity and efficiency for shorter protospacers. Moreover, the results from the genome-wide off-target assessments, compared with other off-target scoring tools, indicate that the statistical mechanics-based approach provides more reliable off-target analyses and sgRNA design. To facilitate the genome engineering applications, a new web-based tool for genome-wide off-target assessment is established.Xiaojun XuDongsheng DuanShi-Jie ChenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-9 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xiaojun Xu Dongsheng Duan Shi-Jie Chen CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment
description	Abstract The CRISPR/Cas9 complex, a bacterial immune response system, has been widely adopted for RNA-guided genome editing and transcription regulation in applications such as targeted genome modification and site-directed mutagenesis. However, the physical basis for its target specificity is not fully understood. In this study, based on a statistical mechanical analysis for the whole ensemble of sgRNA-target complex conformations, we identify a strong correlation between Cas9 cleavage efficiency and the stability of the DNA-RNA (R-loop) complex structures, with a Pearson correlation coefficient ranging from 0.775 to 0.886 for the tested systems. The finding leads to quantitative insights into important experimental results, such as the greater Cas9 tolerance to single-base mismatches in PAM-distal region than to PAM-proximal region and the high specificity and efficiency for shorter protospacers. Moreover, the results from the genome-wide off-target assessments, compared with other off-target scoring tools, indicate that the statistical mechanics-based approach provides more reliable off-target analyses and sgRNA design. To facilitate the genome engineering applications, a new web-based tool for genome-wide off-target assessment is established.
format	article
author	Xiaojun Xu Dongsheng Duan Shi-Jie Chen
author_facet	Xiaojun Xu Dongsheng Duan Shi-Jie Chen
author_sort	Xiaojun Xu
title	CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment
title_short	CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment
title_full	CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment
title_fullStr	CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment
title_full_unstemmed	CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment
title_sort	crispr-cas9 cleavage efficiency correlates strongly with target-sgrna folding stability: from physical mechanism to off-target assessment
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/2ff2b28d1acd498f8b5cb6119d43ad02
work_keys_str_mv	AT xiaojunxu crisprcas9cleavageefficiencycorrelatesstronglywithtargetsgrnafoldingstabilityfromphysicalmechanismtoofftargetassessment AT dongshengduan crisprcas9cleavageefficiencycorrelatesstronglywithtargetsgrnafoldingstabilityfromphysicalmechanismtoofftargetassessment AT shijiechen crisprcas9cleavageefficiencycorrelatesstronglywithtargetsgrnafoldingstabilityfromphysicalmechanismtoofftargetassessment
_version_	1718388570398392320

CRISPR-Cas9 cleavage efficiency correlates strongly with target-sgRNA folding stability: from physical mechanism to off-target assessment

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