The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the natural fusion Cas8-HNH protein for precise target cleavage both in vitro and in human cells. Here, we report multiple cryo-electron microscopy structures of the type I-F Cas8-HNH system at different functional states. The Cas8-HNH Cascade complex adopts an overall G-shaped architecture, with the HNH domain occupying the C-terminal helical bundle domain (HB) of the Cas8 protein in canonical type I systems. The Linker region connecting Cas8-NTD and HNH domains adopts a rigid conformation and interacts with the Cas7.6 subunit, enabling the HNH domain to be in a functional position. The full R-loop formation displaces the HNH domain away from the Cas6 subunit, thus activating the target DNA cleavage. Importantly, our results demonstrate that precise target cleavage is dictated by a C-terminal helix of the HNH domain. Together, our work not only delineates the structural basis for target recognition and activation of the type I-F Cas8-HNH system, but also guides further developments leveraging this system for precise DNA editing.
典型的 I 型 CRISPR-Cas 系统利用 Cas3 核酸酶对目标 DNA 进行过程性降解，而新发现的 I-F 型 CRISPR 变体则利用来自天然融合 Cas8-HNH 蛋白的 HNH 核酸酶结构域，在体外和人体细胞中进行精确的目标切割。在这里，我们报告了 I-F 型 Cas8-HNH 系统在不同功能状态下的多个冷冻电镜结构。Cas8-HNH 级联复合物采用整体 G 型结构，在典型的 I 型系统中，HNH 结构域占据 Cas8 蛋白的 C 端螺旋束结构域（HB）。连接 Cas8-NTD 和 HNH 结构域的 Linker 区域采用刚性构象，并与 Cas7.6 亚基相互作用，使 HNH 结构域处于功能性位置。全 R 环的形成使 HNH 结构域远离 Cas6 亚基，从而激活了目标 DNA 的切割。重要的是，我们的研究结果表明，精确的目标切割是由 HNH 结构域的 C 端螺旋决定的。总之，我们的研究工作不仅阐明了 I-F 型 Cas8-HNH 系统识别和激活靶标的结构基础，还为利用该系统进行精确 DNA 编辑的进一步开发提供了指导。
{"title":"Structural basis for the type I-F Cas8-HNH system.","authors":"Xuzichao Li,Yanan Liu,Jie Han,Lingling Zhang,Zhikun Liu,Lin Wang,Shuqin Zhang,Qian Zhang,Pengyu Fu,Hang Yin,Hongtao Zhu,Heng Zhang","doi":"10.1038/s44318-024-00229-8","DOIUrl":"https://doi.org/10.1038/s44318-024-00229-8","url":null,"abstract":"The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the natural fusion Cas8-HNH protein for precise target cleavage both in vitro and in human cells. Here, we report multiple cryo-electron microscopy structures of the type I-F Cas8-HNH system at different functional states. The Cas8-HNH Cascade complex adopts an overall G-shaped architecture, with the HNH domain occupying the C-terminal helical bundle domain (HB) of the Cas8 protein in canonical type I systems. The Linker region connecting Cas8-NTD and HNH domains adopts a rigid conformation and interacts with the Cas7.6 subunit, enabling the HNH domain to be in a functional position. The full R-loop formation displaces the HNH domain away from the Cas6 subunit, thus activating the target DNA cleavage. Importantly, our results demonstrate that precise target cleavage is dictated by a C-terminal helix of the HNH domain. Together, our work not only delineates the structural basis for target recognition and activation of the type I-F Cas8-HNH system, but also guides further developments leveraging this system for precise DNA editing.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"2015 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142165867","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-19DOI: 10.1038/s44318-023-00017-w
Liyang Du, Qinwei Zhu, Zhonghui Lin
{"title":"Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate","authors":"Liyang Du, Qinwei Zhu, Zhonghui Lin","doi":"10.1038/s44318-023-00017-w","DOIUrl":"https://doi.org/10.1038/s44318-023-00017-w","url":null,"abstract":"","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":" 19","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138961884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-15DOI: 10.1038/s44318-023-00006-z
Jose M Delgado, Logan Wallace Shepard, Sarah W Lamson, Samantha L Liu, Christopher J Shoemaker
{"title":"The ER membrane protein complex restricts mitophagy by controlling BNIP3 turnover","authors":"Jose M Delgado, Logan Wallace Shepard, Sarah W Lamson, Samantha L Liu, Christopher J Shoemaker","doi":"10.1038/s44318-023-00006-z","DOIUrl":"https://doi.org/10.1038/s44318-023-00006-z","url":null,"abstract":"","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"350 3-4","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138996844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-15DOI: 10.1038/s44318-023-00004-1
Joel A. Crossley, W. Allen, Daniel W. Watkins, T. Sabir, S. Radford, Roman Tuma, I. Collinson, Tomas Fessl
{"title":"Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon","authors":"Joel A. Crossley, W. Allen, Daniel W. Watkins, T. Sabir, S. Radford, Roman Tuma, I. Collinson, Tomas Fessl","doi":"10.1038/s44318-023-00004-1","DOIUrl":"https://doi.org/10.1038/s44318-023-00004-1","url":null,"abstract":"","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"12 5","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139000975","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-15DOI: 10.1038/s44318-023-00013-0
Yanli Dong, Zhuoya Yu, Yue Li, Bo Huang, Qinru Bai, Yiwei Gao, Qihao Chen, Na Li, Lingli He, Yan Zhao
{"title":"Structural insight into the allosteric inhibition of human sodium-calcium exchanger NCX1 by XIP and SEA0400","authors":"Yanli Dong, Zhuoya Yu, Yue Li, Bo Huang, Qinru Bai, Yiwei Gao, Qihao Chen, Na Li, Lingli He, Yan Zhao","doi":"10.1038/s44318-023-00013-0","DOIUrl":"https://doi.org/10.1038/s44318-023-00013-0","url":null,"abstract":"","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"27 107","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138998993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-14DOI: 10.1038/s44318-023-00002-3
S. Wong, Aghil Soman, N. Korolev, Wahyu Surya, Qinming Chen, Wayne Shum, John van Noort, L. Nordenskiöld
{"title":"The shelterin component TRF2 mediates columnar stacking of human telomeric chromatin","authors":"S. Wong, Aghil Soman, N. Korolev, Wahyu Surya, Qinming Chen, Wayne Shum, John van Noort, L. Nordenskiöld","doi":"10.1038/s44318-023-00002-3","DOIUrl":"https://doi.org/10.1038/s44318-023-00002-3","url":null,"abstract":"","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"36 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138971482","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-12-11DOI: 10.15252/embj.2023113590
Federico Cocozza, Lorena Martin-Jaular, Lien Lippens, Aurelie Di Cicco, Yago A Arribas, Nicolas Ansart, Florent Dingli, Michael Richard, Louise Merle, Mabel Jouve San Roman, Patrick Poullet, Damarys Loew, Daniel Lévy, An Hendrix, George Kassiotis, Alain Joliot, Mercedes Tkach, Clotilde Théry
Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.
{"title":"Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells","authors":"Federico Cocozza, Lorena Martin-Jaular, Lien Lippens, Aurelie Di Cicco, Yago A Arribas, Nicolas Ansart, Florent Dingli, Michael Richard, Louise Merle, Mabel Jouve San Roman, Patrick Poullet, Damarys Loew, Daniel Lévy, An Hendrix, George Kassiotis, Alain Joliot, Mercedes Tkach, Clotilde Théry","doi":"10.15252/embj.2023113590","DOIUrl":"https://doi.org/10.15252/embj.2023113590","url":null,"abstract":"Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.","PeriodicalId":501009,"journal":{"name":"The EMBO Journal","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138571432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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