Methods in enzymology最新文献_第2页

TaqMan RT-qPCR for tRNA half quantification. TaqMan RT-qPCR用于tRNA半定量。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.010

Megumi Shigematsu, Takuya Kawamura, Yohei Kirino

{"title":"TaqMan RT-qPCR for tRNA half quantification.","authors":"Megumi Shigematsu, Takuya Kawamura, Yohei Kirino","doi":"10.1016/bs.mie.2024.11.010","DOIUrl":"10.1016/bs.mie.2024.11.010","url":null,"abstract":"When quantifying tRNA-derived short non-coding RNAs (sncRNAs), two key considerations must be addressed. First, sequencing analyses have revealed significant heterogeneity in the lengths and terminal sequences of tRNA-derived sncRNAs. Second, within the total RNA fraction, these sncRNAs coexist with more abundant mature tRNAs and their precursors (pre-tRNAs), which share identical sequences with the sncRNAs. While accurate quantification of individual tRNA-derived sncRNAs is crucial for research on these molecules, these two factors make it challenging to achieve with standard RT-qPCR, stem-loop RT-qPCR, and northern blot. We have developed a TaqMan RT-qPCR method that specifically quantifies tRNA half molecules. Here we describe a detailed and recently updated protocol in which an adaptor is ligated to the target tRNA half, and the TaqMan probe targets the boundaries of the tRNA half and adaptor, ensuring specific quantification without cross-reacting with corresponding mature tRNA or pre-tRNA. Our method utilizes only commercially available reagents and is broadly applicable for quantifying tRNA halves and other sncRNAs in diverse samples, including clinical specimens such as human plasma.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"155-170"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11947946/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425830","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

A quick guide to evaluating prime editing efficiency in mammalian cells. 评估哺乳动物细胞初始编辑效率的快速指南。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-31 DOI: 10.1016/bs.mie.2025.01.016

Chengfang Liu, Sifan Cheng, Junjie Zhu, Lina Zhou, Jia Chen

{"title":"A quick guide to evaluating prime editing efficiency in mammalian cells.","authors":"Chengfang Liu, Sifan Cheng, Junjie Zhu, Lina Zhou, Jia Chen","doi":"10.1016/bs.mie.2025.01.016","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.016","url":null,"abstract":"According to the Clinvar database, modeling the diseases associated with pathogenic mutations requires the installation of base substitutions, small insertions or deletions. Prime editor (PE) was recently developed to precisely install any base substitutions and/or small insertions/deletions (indels) in mammalian cells and animals without requiring DSBs or donor DNA templates. PE also offers greater editing and targeting flexibility compared to other precision CRISPR editing methods because the versatile editing information is encoded in the reverse-transcription template of its prime editing guide RNA. However, optimal PE system selection and experimental design can be complex, and there are various factors that can affect PE efficiency. This chapter serves as a rapid entry-level guideline for the application of PE, providing an experimental framework for using PE at a specific genomic locus. RUNX1 was selected as a representative target site to illustrate the detailed methodology for constructing PE plasmids and the process of transfecting these plasmids into 293FT cells. We further examined the efficiency of PE-mediated genome editing in mammalian cells by using next-generation sequencing.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"419-436"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692734","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

Nanopore sequencing to detect A-to-I editing sites. 纳米孔测序检测A-to-I编辑位点。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.028

Jia Wei Joel Heng, Meng How Tan

引用次数: 0

Restoration of G to A mutated transcripts using the MS2-ADAR1 system. 利用MS2-ADAR1系统恢复G到A突变转录本。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-05 DOI: 10.1016/bs.mie.2024.11.031

Sonali Bhakta, Toshifumi Tsukahara

{"title":"Restoration of G to A mutated transcripts using the MS2-ADAR1 system.","authors":"Sonali Bhakta, Toshifumi Tsukahara","doi":"10.1016/bs.mie.2024.11.031","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.031","url":null,"abstract":"Site-directed RNA editing (SDRE) holds significant promise for treating genetic disorders resulting from point mutations. Gene therapy, for common genetic illnesses is becoming more popular and, although viable treatments for genetic disorders are scarce, stop codon mutation-related conditions may benefit from gene editing. Effective SDRE generally depends on introducing many guideRNA molecules relative to the target gene; however, large ratios cannot be achieved in the context of gene therapy applications. Gene-encoded information can be altered, and functionally diverse proteins produced from a single gene by restoration of point-mutated RNA molecules using SDRE. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme, that can specifically convert adenosine (A) residues to inosines (I), which are translated as guanosine (G). MS2 system along with ADAR1 deaminase domain can target a particular A and repair G to A mutations. In this study, we used the RNA binding MS2 coat protein fused with the ADAR1 deaminase domain controlled by the CMV promoter, and a 19 bp guide RNA (complementary to the target mRNA sequence) engineered with 6 × MS2 stem-loops downstream or 1 × MS2 stem-loop (double MS2) on either side, controlled by the U6 promoter. When the EGFP TGG codon (tryptophan) was altered to an amber (TAG), opal (TGA), or ochre (TAA) stop codon, the modified ADAR1 deaminase domain could convert A-to-I (G) at the edited sites. It is anticipated that successful establishment of this technique will result in a new era in gene therapy, allowing remarkably efficient gene repair, even in vivo.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"229-240"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052993","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

Editing specificity of ADAR isoforms. ADAR同工型的编辑特异性。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.021

Cornelia Vesely, Michael F Jantsch

{"title":"Editing specificity of ADAR isoforms.","authors":"Cornelia Vesely, Michael F Jantsch","doi":"10.1016/bs.mie.2024.11.021","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.021","url":null,"abstract":"Adenosine to inosine deaminases acting on RNA (ADARs) enzymes are found in all metazoa. Their sequence and protein organization is conserved but also shows distinct differences. Moreover, the number of ADAR genes differs between organisms, ranging from one in flies to three in mammals. The distinct isoforms of ADARs and their specific roles determine the complexity of A-to-I RNA editing, its regulation and the versatility of these enzymes. Understanding the different isoform-specific functions and targets will provide a deeper understanding of the diverse biological processes influenced by ADARs, either through ADAR editing of dsRNAs or the interaction with RNAs and proteins. The detailed identification and assigning of isoform-specific targets is a crucial step towards our understanding of functional differences amongst ADAR isoforms and will help us to understand their individual implications for health and disease. This chapter delves into unique characteristics and functional implications of ADAR isoforms. We describe the ectopic overexpression in editing free cells and the use of RNA immunoprecipitation coupled with sequencing to determine isoform-specific interactions with RNAs and their editing sites. Additionally, we discuss new challenges in editing detection by different ADARs in the context of other modifications and provide ideas for potentially better methods to determine the \"true editome\".","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"77-98"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053006","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

Aptazyme-directed A-to-I RNA editing. 适配体酶导向的A-to-I RNA编辑。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.022

Xilei Ai, Zhuo Tang

{"title":"Aptazyme-directed A-to-I RNA editing.","authors":"Xilei Ai, Zhuo Tang","doi":"10.1016/bs.mie.2024.11.022","DOIUrl":"10.1016/bs.mie.2024.11.022","url":null,"abstract":"As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-λN-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"267-283"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052979","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

En masse evaluation of RNA guides (EMERGe) for ADARs. RNA指南（EMERGe）对ADARs的整体评价。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-02 DOI: 10.1016/bs.mie.2024.11.030

Prince J Salvador, Natalie M Dugan, Randall Ouye, Peter A Beal

{"title":"En masse evaluation of RNA guides (EMERGe) for ADARs.","authors":"Prince J Salvador, Natalie M Dugan, Randall Ouye, Peter A Beal","doi":"10.1016/bs.mie.2024.11.030","DOIUrl":"10.1016/bs.mie.2024.11.030","url":null,"abstract":"Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in duplex RNA, and through the delivery of guide RNAs, can be directed to edit specific adenosine sites. As ADARs are endogenously expressed in humans, their editing capacities hold therapeutic potential and allow us to target disease-relevant sequences in RNA through the rationale design of guide RNAs. However, current design principles are not suitable for difficult-to-edit target sites, posing challenges to unlocking the full therapeutic potential of this approach. This chapter discusses how we circumvent this barrier through an in vitro screening method, En Masse Evaluation of RNA Guides (EMERGe), which enables comprehensive screening of ADAR substrate libraries and facilitates the identification of editing-enabling guide strands for specific adenosines. From library generation and screening to next generation sequencing (NGS) data analysis to verification experiments, we describe how a sequence of interest can be identified through this high-throughput screening method. Furthermore, we discuss downstream applications of selected guide sequences, challenges in maximizing library coverage, and potential to couple the screen with machine learning or deep learning models.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"131-152"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053011","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

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":"https://doi.org/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

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。

4区生物学

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":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

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