Methods in enzymology最新文献_第7页

Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2. CRISPR-Cas12a2的纯化及体内、无细胞和体外鉴定。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-07 DOI: 10.1016/bs.mie.2025.01.032

Friso T Schut, Thomson Hallmark, Oleg Dmytrenko, Ryan N Jackson, Chase L Beisel

{"title":"Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2.","authors":"Friso T Schut, Thomson Hallmark, Oleg Dmytrenko, Ryan N Jackson, Chase L Beisel","doi":"10.1016/bs.mie.2025.01.032","DOIUrl":"10.1016/bs.mie.2025.01.032","url":null,"abstract":"The CRISPR-associated (Cas) nuclease Cas12a2 from Sulfuricurvum sp. PC08-66 (SuCas12a2) binds RNA targets with a complementary guide (g)RNA. Target RNA binding causes a major conformational rearrangement in Cas12a2 that activates a RuvC nuclease domain to collaterally cleave RNA, ssDNA and dsDNA, arresting growth and providing population-level immunity. Here, we report in vivo, cell-free, and in vitro methods to characterize the collateral cleavage activity of SuCas12a2 as well as a procedure for gRNA design. As part of the in vivo methods, we describe how to capture growth arrest through plasmid interference and induction of an SOS DNA damage response in the bacterium Escherichia coli. We further apply cell-free transcription-translation to affirm collateral cleavage activity triggered by an expressed RNA target. Finally, as part of the in vitro methods, we describe how to purify active nuclease and subsequently conduct biochemical cleavage assays. In total, the outlined methods should accelerate the exploration of SuCas12a2 and other related Cas nucleases, revealing new features of CRISPR biology and helping develop new CRISPR technologies for molecular diagnostics and other applications.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"143-181"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692750","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

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。

4区生物学

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

Prime editing in bacteria with BacPE. 带BacPE的细菌启动编辑。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-20 DOI: 10.1016/bs.mie.2025.01.026

Hongyuan Zhang, Quanjiang Ji

引用次数: 0

In vitro functional analysis of plant tDRs. 植物tDRs的离体功能分析。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-03 DOI: 10.1016/bs.mie.2024.11.011

Christina Berrissou, Laurence Drouard

引用次数: 0

Genetic analyses of Myc and hypusine circuits in tumorigenesis. 肿瘤发生中Myc和hypusine回路的遗传分析。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-21 DOI: 10.1016/bs.mie.2025.02.005

Shima Nakanishi, John L Cleveland

{"title":"Genetic analyses of Myc and hypusine circuits in tumorigenesis.","authors":"Shima Nakanishi, John L Cleveland","doi":"10.1016/bs.mie.2025.02.005","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.02.005","url":null,"abstract":"A prominent metabolic pathway induced by MYC family oncoproteins in cancer is the polyamine-hypusine circuit, which post-translationally modifies a specific lysine residue of eukaryotic translation initiation factor 5 A (eIF5A) with a unique amino acid coined hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. This modification occurs in a two-step process, whereby the aminobutyl group of the polyamine spermidine is covalently linked to lysine-50 of eIF5A via deoxyhypusine synthase (DHPS) to form the intermediate deoxyhypusinated eIF5A, which is subsequently hydoxylated by deoxyhypusine hydroxylase (DOHH) to form the fully mature eIF5AHyp. As a result, eIF5AHyp is elevated in MYC-driven cancers. Recently it has become evident that eIF5AHyp (i) plays key roles in the development, progression and maintenance of tumors; and (ii) eIF5AHyp functions are often tissue/cell context-specific. Thus, it is important to mechanistically assess how eIF5AHyp affects normal cells and tumorigenesis using suitable in vivo and ex vivo models. In this chapter, we describe the methods used in our laboratory to assess the effects of MYC-polyamine-hypusine axis on the development and maintenance of MYC-driven B-cell lymphoma. The goals of this chapter are twofold. First, we discuss genetic and cell biological approaches that can be applied to assess roles of eIF5AHyp on lymphoma and normal B cell development. Second, we discuss methods that can be used to assess the roles of eIF5AHyp on the growth and maintenance of lymphoma. Collectively, these approaches provide a template that can be applied to evaluate roles of any putative regulator of the development and/or maintenance of lymphoma.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"1-17"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094217","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

Monitoring ODC activity and polyamines in Bachmann-Bupp syndrome patient biological samples. 监测巴赫曼-布普综合征患者生物样本中ODC活性和多胺。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-26 DOI: 10.1016/bs.mie.2025.01.071

Chad R Schultz, Elizabeth A VanSickle, Caleb P Bupp, André S Bachmann

{"title":"Monitoring ODC activity and polyamines in Bachmann-Bupp syndrome patient biological samples.","authors":"Chad R Schultz, Elizabeth A VanSickle, Caleb P Bupp, André S Bachmann","doi":"10.1016/bs.mie.2025.01.071","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.071","url":null,"abstract":"Polyamines are aliphatic molecules that include putrescine, spermidine, and spermine. Polyamines are present in most living organisms including humans. These positively charged molecules play important roles in cell physiology and pathology by contributing to embryonic cell development, regulation of cell division and, if overproduced, the stimulation of cancer cell proliferation and tumorigenesis. We recently discovered Bachmann-Bupp Syndrome (BABS); a rare neurodevelopmental disorder linked to de novo mutations in the ornithine decarboxylase 1 (ODC1) gene. ODC1 gene mutations that are linked to BABS always produce C-terminally truncated versions of the enzyme ornithine decarboxylase (ODC). These shortened ODC proteins remain enzymatically active and are not cleared by the proteasome, therefore leading to ODC protein accumulation in cells. ODC is a key enzyme of polyamine biosynthesis by converting ornithine to putrescine, and if accumulated, can lead to high putrescine levels in human cells including red blood cells (RBCs) and primary dermal fibroblasts. Here we describe how to quantitatively measure ODC enzymatic activity and the polyamines by a radiolabeled 14C-ornithine assay and by reverse phase (RP)-HPLC, respectively. While these methods have been developed decades ago, many publications provide incomplete protocols with omission of experimental details, which inadvertently can lead to mistakes, inconclusive results, and failed experiments. There is a growing number of laboratories that have become interested in exploring polyamines (in part due to metabolomics analyses in human health-related studies). The detailed protocols of this chapter provide step-by-step guidance detailing how to measure ODC activity and polyamines in human RBCs.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"257-270"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094311","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