Pub Date : 2026-03-25DOI: 10.1038/s41594-026-01761-3
Feizuo Wang, Ruochen Guo, Senfeng Zhang, Yinuo Cui, Junlan Wang, Tao Hu, Kunming Liu, Qi Wang, Yao Liu, Ki Hyun Nam, Ziqing Winston Zhao, Quanquan Ji, Xin Xu, Ercheng Wang, Youyuan Zhu, Yao Yang, Min Luo, Peixiang Ma, Shengsheng Ma, Chunlong Xu, Chunyi Hu
IscB, a compact Cas9 ancestor from the obligate mobile element guided activity system, has attracted growing interest as a programmable genome editor because of its small size and therapeutic delivery potential. Despite its promise, structural insights into IscB’s regulation remain limited, with only a target-bound R-loop structure previously reported. Here, we present the structural trajectory of an engineered IscB, capturing its transition from a resting state to activation. Using cryo-electron microscopy, we resolve four high-resolution structures: the apo resting state, two intermediate complexes with 6-nt and 10-nt guide–target pairing and a fully paired 16-nt primed cleavage state. These structures uncover a dual inactivation mechanism mediated by RNA lids; the ωRNA lid blocks HNH domain access, while the guide RNA lid occludes the RuvC active site. As guide–target pairing progresses, the guide RNA undergoes a stepwise displacement, mimicking a ‘car pedal’ motion that triggers activation at 11-nt pairing. The HNH domain also contributes to R-loop stabilization through a positively charged R-wedge motif and undergoes a ~90° activation-driven rotation mediated by two hinge regions. In variants IscBHig1 and IscBHig2, engineering these hinge motifs to enhance conformational flexibility notably improved genome-editing efficiency in cells. In summary, our study reveals the molecular basis underlying IscB autoinhibition and activation, identifies previously uncharacterized regulatory features and establishes hinge elements as a target region for engineering compact, efficient genome editors.
{"title":"Structural insight into IscB’s RNA-lid-based inactivation mechanism","authors":"Feizuo Wang, Ruochen Guo, Senfeng Zhang, Yinuo Cui, Junlan Wang, Tao Hu, Kunming Liu, Qi Wang, Yao Liu, Ki Hyun Nam, Ziqing Winston Zhao, Quanquan Ji, Xin Xu, Ercheng Wang, Youyuan Zhu, Yao Yang, Min Luo, Peixiang Ma, Shengsheng Ma, Chunlong Xu, Chunyi Hu","doi":"10.1038/s41594-026-01761-3","DOIUrl":"https://doi.org/10.1038/s41594-026-01761-3","url":null,"abstract":"IscB, a compact Cas9 ancestor from the obligate mobile element guided activity system, has attracted growing interest as a programmable genome editor because of its small size and therapeutic delivery potential. Despite its promise, structural insights into IscB’s regulation remain limited, with only a target-bound R-loop structure previously reported. Here, we present the structural trajectory of an engineered IscB, capturing its transition from a resting state to activation. Using cryo-electron microscopy, we resolve four high-resolution structures: the apo resting state, two intermediate complexes with 6-nt and 10-nt guide–target pairing and a fully paired 16-nt primed cleavage state. These structures uncover a dual inactivation mechanism mediated by RNA lids; the ωRNA lid blocks HNH domain access, while the guide RNA lid occludes the RuvC active site. As guide–target pairing progresses, the guide RNA undergoes a stepwise displacement, mimicking a ‘car pedal’ motion that triggers activation at 11-nt pairing. The HNH domain also contributes to R-loop stabilization through a positively charged R-wedge motif and undergoes a ~90° activation-driven rotation mediated by two hinge regions. In variants IscBHig1 and IscBHig2, engineering these hinge motifs to enhance conformational flexibility notably improved genome-editing efficiency in cells. In summary, our study reveals the molecular basis underlying IscB autoinhibition and activation, identifies previously uncharacterized regulatory features and establishes hinge elements as a target region for engineering compact, efficient genome editors.","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147506133","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 : 2026-03-18DOI: 10.1038/s41594-026-01753-3
Rong Zheng,Zhike Lu,Rongwei Wei,Young-Cheul Shin,Jiang Du,Qingfeng Zhang,Jianbo Li,Xiaoqi Wang,Yi Wei,Botao Liu,Yang Chen,Lihong Ding,Heng Zhang,Hui Chen,Jing Huang,Lijia Ma
Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.
{"title":"Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions.","authors":"Rong Zheng,Zhike Lu,Rongwei Wei,Young-Cheul Shin,Jiang Du,Qingfeng Zhang,Jianbo Li,Xiaoqi Wang,Yi Wei,Botao Liu,Yang Chen,Lihong Ding,Heng Zhang,Hui Chen,Jing Huang,Lijia Ma","doi":"10.1038/s41594-026-01753-3","DOIUrl":"https://doi.org/10.1038/s41594-026-01753-3","url":null,"abstract":"Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147478549","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 : 2026-03-17DOI: 10.1038/s41594-026-01771-1
Toshifumi Inada
Ribosome-associated quality control (RQC) safeguards translation by detecting and resolving collided ribosomes and triaging their nascent chains. This Review outlines mechanisms, crosstalk and disease implications of RQC cascades and presents RQC as a 'first responder' that prevents escalation to global stress responses and provides protection against proteostasis collapse.
{"title":"Ribosome-associated quality control and related mechanisms.","authors":"Toshifumi Inada","doi":"10.1038/s41594-026-01771-1","DOIUrl":"https://doi.org/10.1038/s41594-026-01771-1","url":null,"abstract":"Ribosome-associated quality control (RQC) safeguards translation by detecting and resolving collided ribosomes and triaging their nascent chains. This Review outlines mechanisms, crosstalk and disease implications of RQC cascades and presents RQC as a 'first responder' that prevents escalation to global stress responses and provides protection against proteostasis collapse.","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147471388","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 : 2026-03-13DOI: 10.1038/s41594-026-01759-x
Kirandeep K. Deol, Cynthia A. Harris, Sydney J. Tomlinson, Colin J. Delaney, Amr Al-Farhan, Alyssa J. Mathiowetz, Cody E. Doubravsky, Derek A. Pratt, James A. Olzmann
Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR–Cas9 screens using a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase and flavin adenine dinucleotide (FAD) synthase, enzymes that are essential for synthesizing FAD from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for both FSP1 activity and stability. FAD deficiency and mutations blocking FSP1–FAD binding triggered FSP1 degradation through a ubiquitin–proteasome pathway involving the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance, with implications for therapeutic strategies targeting FSP1 in cancer.
{"title":"Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis","authors":"Kirandeep K. Deol, Cynthia A. Harris, Sydney J. Tomlinson, Colin J. Delaney, Amr Al-Farhan, Alyssa J. Mathiowetz, Cody E. Doubravsky, Derek A. Pratt, James A. Olzmann","doi":"10.1038/s41594-026-01759-x","DOIUrl":"https://doi.org/10.1038/s41594-026-01759-x","url":null,"abstract":"Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR–Cas9 screens using a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase and flavin adenine dinucleotide (FAD) synthase, enzymes that are essential for synthesizing FAD from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for both FSP1 activity and stability. FAD deficiency and mutations blocking FSP1–FAD binding triggered FSP1 degradation through a ubiquitin–proteasome pathway involving the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance, with implications for therapeutic strategies targeting FSP1 in cancer.","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147454761","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 : 2026-03-11DOI: 10.1038/s41594-026-01767-x
{"title":"PADI6 binds UBE2D and UHRF1 to control protein ubiquitination in oocytes.","authors":"","doi":"10.1038/s41594-026-01767-x","DOIUrl":"https://doi.org/10.1038/s41594-026-01767-x","url":null,"abstract":"","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":"315 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147394016","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 : 2026-03-06DOI: 10.1038/s41594-026-01750-6
{"title":"Charting the 3D regulatory landscape of sex determination with geostatistics.","authors":"","doi":"10.1038/s41594-026-01750-6","DOIUrl":"https://doi.org/10.1038/s41594-026-01750-6","url":null,"abstract":"","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":"29 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147368444","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 : 2026-03-06DOI: 10.1038/s41594-026-01773-z
Edith Heard,Gitte Neubauer
{"title":"Factors for a successful partnership between academia and industry.","authors":"Edith Heard,Gitte Neubauer","doi":"10.1038/s41594-026-01773-z","DOIUrl":"https://doi.org/10.1038/s41594-026-01773-z","url":null,"abstract":"","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":"6 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147368443","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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