Methods in enzymology最新文献_第6页

Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins. 测定IV型CRISPR核糖核蛋白复合物和辅助蛋白的生化功能。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-11 DOI: 10.1016/bs.mie.2025.01.039

Andrew A Williams, Olivine Redman, Hannah Domgaard, Matthew J Armbrust, Ryan N Jackson

引用次数: 0

CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells. CRISPRoff表观基因组编辑用于人类细胞系和原代T细胞的可编程基因沉默。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.010

Rithu K Pattali, Izaiah J Ornelas, Carolyn D Nguyen, Da Xu, Nikita S Divekar, NunezJames K Nuñez

{"title":"CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells.","authors":"Rithu K Pattali, Izaiah J Ornelas, Carolyn D Nguyen, Da Xu, Nikita S Divekar, NunezJames K Nuñez","doi":"10.1016/bs.mie.2025.01.010","DOIUrl":"10.1016/bs.mie.2025.01.010","url":null,"abstract":"The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"517-551"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12052198/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Fluorescent shift assay for APOBEC-mediated RNA editing. apobecc介导的RNA编辑的荧光位移试验。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-10 DOI: 10.1016/bs.mie.2024.12.002

Shanshan Wang, Benjamin Fixman, Xiaojiang S Chen

{"title":"Fluorescent shift assay for APOBEC-mediated RNA editing.","authors":"Shanshan Wang, Benjamin Fixman, Xiaojiang S Chen","doi":"10.1016/bs.mie.2024.12.002","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.12.002","url":null,"abstract":"Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is involved in diverse biological processes. The APOBEC deaminase family acts in various cellular processes mostly through inducing C-to-U mutation in single-stranded RNA (or DNA). However, comparing the activity of different RNA editing enzymes to one another is difficult due to the limited number of systems that can provide direct and efficient readout. In this report, a system in which RNA editing directly prompts a change in the subcellular localization of a modified eGFP structure is described in detail. This approach allows us to compare relative fluorescence intensity based on the RNA editing level. When observed through a fluorescence detection system, like a scanning confocal microscope, the cellular nucleus can be readily identified using a DNA-binding stain, such as DAPI or Hoechst, so that the accurate calculation of the ratio of nuclear to cytosolic eGFP intensity can be applied for an individual cell. This method provides a useful and flexible tool to examine and quantify RNA editing activity within cells, and it is not only limited to APOBEC proteins, but can also be applied more generally to other RNA editing enzymatic assays.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"1-14"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144002689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Gene replacement therapy to restore polyamine metabolism in a Snyder-Robinson syndrome mouse model. 基因替代疗法恢复Snyder-Robinson综合征小鼠模型中的多胺代谢。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-16 DOI: 10.1016/bs.mie.2025.01.068

Oluwaseun Akinyele, Krystal B Tran, Marie A Johnson, Dwi U Kemaladewi

{"title":"Gene replacement therapy to restore polyamine metabolism in a Snyder-Robinson syndrome mouse model.","authors":"Oluwaseun Akinyele, Krystal B Tran, Marie A Johnson, Dwi U Kemaladewi","doi":"10.1016/bs.mie.2025.01.068","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.068","url":null,"abstract":"Polyamines, including putrescine, spermidine, and spermine, are organic cations essential for cell growth, proliferation, and tissue regeneration. Their levels are tightly regulated by a set of enzymes controlling their biosynthesis, catabolism, and interconversion. Dysregulation of polyamine metabolism is associated with a group of rare genetic neurodevelopmental disorders collectively known as \"polyaminopathies\", including Snyder-Robinson Syndrome (SRS). SRS is an X-linked recessive disorder caused by mutations in the SMS gene, which encodes the spermine synthase enzyme. The lack of spermine synthase leads to aberrant polyamine levels and neurological impairments, as observed in patients and animal models. Currently, there are no available treatment options for SRS. Due to its monogenic nature, SRS is an excellent candidate for gene replacement therapy. The recent success of Zolgensma in treating children with Spinal Muscular Atrophy and the establishment of Platform Vector Gene Therapy (Pave-GT) initiative at the National Institute of Health (NIH) offer a framework to adapt-and-apply the same gene delivery system for multiple rare disease gene therapies. This chapter outlines strategies for delivering a functional copy of the SMS gene using an adeno-associated viral (AAV) vector, as well as methods to evaluate the molecular efficacy of this approach in an SRS mouse model. Our ultimate goal is to establish a versatile platform for genetic interventions targeting SRS and other polyaminopathies.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"271-292"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094212","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

NMR spectroscopy: An invaluable tool to identify lignin structural modifications induced by oxidative enzymes. 核磁共振波谱：鉴定氧化酶诱导的木质素结构修饰的宝贵工具。

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Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-26 DOI: 10.1016/bs.mie.2025.02.003

Nicolò Pajer, Umberto Danelon, Claudia Crestini

引用次数: 0

Defining the genome-wide mutagenic impact of APOBEC3 enzymes. 定义APOBEC3酶的全基因组诱变作用。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-18 DOI: 10.1016/bs.mie.2024.12.003

Eszter Németh, Rachel A DeWeerd, Abby M Green, Dávid Szüts

引用次数: 0

Digitalization of biocatalysis: Best practices to research data management. 生物催化的数字化：研究数据管理的最佳实践。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-13 DOI: 10.1016/bs.mie.2025.01.040

Torsten Giess, Jürgen Pleiss

{"title":"Digitalization of biocatalysis: Best practices to research data management.","authors":"Torsten Giess, Jürgen Pleiss","doi":"10.1016/bs.mie.2025.01.040","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.040","url":null,"abstract":"The digitalization of biocatalysis presents significant opportunities for advancing research by improving data management, fostering transparency, and enabling more efficient, reproducible experiments. However, this transformation brings challenges, particularly in standardizing and sharing data across diverse platforms and laboratory settings. Managing experimental data and metadata in structured, machine-readable formats is fundamental for integrating automation, while mechanistic modeling and artificial intelligence applications further benefit from well-curated datasets. Creating sustainable, reusable software is also key to the long-term success of biocatalysis projects. Yet, efficient data acquisition remains limited by the lack of universally accepted data formats for analytical instruments. To address these barriers, the best practices presented here focus on optimizing biocatalysis workflows for the FAIR (Findable, Accessible, Interoperable, Reusable) data principles. This includes adopting standardized data exchange formats and sharing reproducible datasets in public repositories, thus enhancing interoperability and reusability. By following these guidelines, researchers can contribute to the digitalization of biocatalysis, facilitating the knowledge sharing and data reuse necessary to support the transition of biocatalysis into a more data-driven field.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"19-43"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143971141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Global quantification of off-target activity by base editors. 碱基编辑器对脱靶活动的全局量化。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-18 DOI: 10.1016/bs.mie.2024.11.036

Michelle Eidelman, Eli Eisenberg, Erez Y Levanon

{"title":"Global quantification of off-target activity by base editors.","authors":"Michelle Eidelman, Eli Eisenberg, Erez Y Levanon","doi":"10.1016/bs.mie.2024.11.036","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.036","url":null,"abstract":"Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"255-270"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144008355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Preface. 前言。

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Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00171-5

Nina F Papavasiliou, Riccardo Pecori

引用次数: 0

Functional characterization of tRNA-derived small RNAs in stem cells. 干细胞中trna衍生小rna的功能表征。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.015

Sowndarya Muthukumar, Silvia Tucciarone, Alexandre André Germanos, Cristian Bellodi

{"title":"Functional characterization of tRNA-derived small RNAs in stem cells.","authors":"Sowndarya Muthukumar, Silvia Tucciarone, Alexandre André Germanos, Cristian Bellodi","doi":"10.1016/bs.mie.2024.11.015","DOIUrl":"10.1016/bs.mie.2024.11.015","url":null,"abstract":"Transfer RNA (tRNA)-derived RNAs (tDRs) are abundant small RNAs with emerging roles in development and tumorigenesis. Increasing evidence indicates that tDRs regulate stem cell homeostasis and differentiation, often altered in disease, highlighting the importance of fully characterizing their role in stem cell biology. Multiple studies point to protein synthesis as a crucial target of tDR-mediated control of different stem cell types. Translation is a highly regulated process that integrates various input signals from cell-intrinsic and -extrinsic cues. Notably, tDRs largely impact translation initiation and ribosome biogenesis, driving critical adaptations of the stem cell proteome and balancing dynamic transitions between self-renewal, proliferation, and cell-fate trajectories. Hematopoietic stem cells (HSCs) give rise to all circulating blood cells and exhibit exquisite sensitivity to tDR-mediated translation control impacting HSC homeostasis and differentiation. Significantly, defects in tDR levels and processing may drive malignant phenotypes in HSCs by supporting aberrant proteomic programs associated with leukemia transformation. While sequencing technologies have dramatically improved tDR detection and quantification, the specific mechanisms by which tDRs impact cellular phenotypes remain incompletely understood. With this increased resolution, further studies will lead to novel insights on the roles of tDRs in crucial stem cell phenotypes. In this chapter, we showcase useful protocols to characterize the molecular functions of tDRs in stem cell populations. We include methods to quantify the effects of tDR on protein synthesis and stem cell proliferation and differentiation. Finally, we highlight in vivo techniques to measure tDR impact on HSC engraftment potential in xenograft models.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"261-282"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425679","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0