Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6

CRISPR-Cas systems are prokaryotic adaptive immunity against invading phages and plasmids. Phages have evolved diverse protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, to neutralize this CRISPR machinery. In response, bacteria have co-evolved Cas variants to escape phage’...

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Autores principales:	Xinyi Li, Chengxiang Wang, Ting Peng, Zongtao Chai, Duan Ni, Yaqin Liu, Jian Zhang, Ting Chen, Shaoyong Lu
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	CRISPR-Cas Anti-CRISPR proteins Molecular dynamics simulations Conformational dynamics Gene editing Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/09b8e01bef4b459c82c24cad411db231
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spelling	oai:doaj.org-article:09b8e01bef4b459c82c24cad411db2312021-11-26T04:26:45ZAtomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA62001-037010.1016/j.csbj.2021.11.010https://doaj.org/article/09b8e01bef4b459c82c24cad411db2312021-01-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S200103702100475Xhttps://doaj.org/toc/2001-0370CRISPR-Cas systems are prokaryotic adaptive immunity against invading phages and plasmids. Phages have evolved diverse protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, to neutralize this CRISPR machinery. In response, bacteria have co-evolved Cas variants to escape phage’s anti-CRISPR strategies, called anti-anti-CRISPR systems. Here we explore the anti-CRISPR allosteric inhibition and anti-anti-CRISPR rescue mechanisms between Streptococcus thermophilus Cas9 (St1Cas9) and the anti-CRISPR protein AcrIIA6 at the atomic level, by generating mutants of key residues in St1Cas9. Extensive unbiased molecular dynamics simulations show that the functional motions of St1Cas9 in the presence of AcrIIA6 differ substantially from those of St1Cas9 alone. AcrIIA6 binding triggers a shift of St1Cas9 conformational ensemble towards a less catalytically competent state; this state significantly compromises protospacer adjacent motif (PAM) recognition and nuclease activity by altering interdependently conformational dynamics and allosteric signals among nuclease domains, PAM-interacting (PI) regions, and AcrIIA6 binding motifs. Via in vitro DNA cleavage assays, we further elucidate the rescue mechanism of efficiently escaping AcrIIA6 inhibition harboring St1Cas9 triple mutations (G993K/K1008M/K1010E) in the PI domain and identify the evolutionary landscape of such mutational escape within species. Our results provide mechanistic insights into Acr proteins as natural brakes for the CRISPR-Cas systems and a promising potential for the design of allosteric Acr peptidomimetics.Xinyi LiChengxiang WangTing PengZongtao ChaiDuan NiYaqin LiuJian ZhangTing ChenShaoyong LuElsevierarticleCRISPR-CasAnti-CRISPR proteinsMolecular dynamics simulationsConformational dynamicsGene editingBiotechnologyTP248.13-248.65ENComputational and Structural Biotechnology Journal, Vol 19, Iss , Pp 6108-6124 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR-Cas Anti-CRISPR proteins Molecular dynamics simulations Conformational dynamics Gene editing Biotechnology TP248.13-248.65
spellingShingle	CRISPR-Cas Anti-CRISPR proteins Molecular dynamics simulations Conformational dynamics Gene editing Biotechnology TP248.13-248.65 Xinyi Li Chengxiang Wang Ting Peng Zongtao Chai Duan Ni Yaqin Liu Jian Zhang Ting Chen Shaoyong Lu Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6
description	CRISPR-Cas systems are prokaryotic adaptive immunity against invading phages and plasmids. Phages have evolved diverse protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, to neutralize this CRISPR machinery. In response, bacteria have co-evolved Cas variants to escape phage’s anti-CRISPR strategies, called anti-anti-CRISPR systems. Here we explore the anti-CRISPR allosteric inhibition and anti-anti-CRISPR rescue mechanisms between Streptococcus thermophilus Cas9 (St1Cas9) and the anti-CRISPR protein AcrIIA6 at the atomic level, by generating mutants of key residues in St1Cas9. Extensive unbiased molecular dynamics simulations show that the functional motions of St1Cas9 in the presence of AcrIIA6 differ substantially from those of St1Cas9 alone. AcrIIA6 binding triggers a shift of St1Cas9 conformational ensemble towards a less catalytically competent state; this state significantly compromises protospacer adjacent motif (PAM) recognition and nuclease activity by altering interdependently conformational dynamics and allosteric signals among nuclease domains, PAM-interacting (PI) regions, and AcrIIA6 binding motifs. Via in vitro DNA cleavage assays, we further elucidate the rescue mechanism of efficiently escaping AcrIIA6 inhibition harboring St1Cas9 triple mutations (G993K/K1008M/K1010E) in the PI domain and identify the evolutionary landscape of such mutational escape within species. Our results provide mechanistic insights into Acr proteins as natural brakes for the CRISPR-Cas systems and a promising potential for the design of allosteric Acr peptidomimetics.
format	article
author	Xinyi Li Chengxiang Wang Ting Peng Zongtao Chai Duan Ni Yaqin Liu Jian Zhang Ting Chen Shaoyong Lu
author_facet	Xinyi Li Chengxiang Wang Ting Peng Zongtao Chai Duan Ni Yaqin Liu Jian Zhang Ting Chen Shaoyong Lu
author_sort	Xinyi Li
title	Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6
title_short	Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6
title_full	Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6
title_fullStr	Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6
title_full_unstemmed	Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6
title_sort	atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of streptococcus thermophilus cas9 by the anti-crispr protein acriia6
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/09b8e01bef4b459c82c24cad411db231
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Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6

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