Methods in enzymology最新文献_第2页

Restoration of G to A mutated transcripts using the MS2-ADAR1 system.

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.

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

Using CRISPR for viral nucleic acid detection.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-01 DOI: 10.1016/bs.mie.2025.01.031

Maaike S Grimm, Cameron Myhrvold

{"title":"Using CRISPR for viral nucleic acid detection.","authors":"Maaike S Grimm, Cameron Myhrvold","doi":"10.1016/bs.mie.2025.01.031","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.031","url":null,"abstract":"Pathogenic microorganisms, such as viruses, have threatened human health and will continue to contribute to future epidemics and pandemics, highlighting the importance of developing effective diagnostics. To contain viral outbreaks within populations, fast and early diagnosis of infected individuals is essential. Although current standard methods are highly sensitive and specific, like RT-qPCR, some can have slow turnaround times, which can hinder the prevention of viral transmission. The discovery of CRISPR-Cas systems in bacteria and archaea initially revolutionized the world of genome editing. Intriguingly, CRISPR-Cas enzymes also have the ability to detect nucleic acids with high sensitivity and specificity, which sparked the interest of researchers to also explore their potential in diagnosis of viral pathogens. In particular, the CRISPR-Cas13 system has been used as a tool for detecting viral nucleic acids. Cas13's capability to detect both target RNA and non-specific RNAs has led to the development of detection methods that leverage these characteristics through designing specific detection read-outs. Optimization of viral sample collection, amplification steps and the detection process within the Cas13 detection workflow has resulted in assays with high sensitivity, rapid turnaround times and the capacity for large-scale implementation. This review focuses on the significant innovations of various CRISPR-Cas13-based viral nucleic acid detection methods, comparing their strengths and weaknesses while highlighting Cas13's great potential as a tool for viral diagnostics.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"245-275"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692834","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

A quick guide to evaluating prime editing efficiency in mammalian cells.

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

TaqMan RT-qPCR for tRNA half quantification.

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

En masse evaluation of RNA guides (EMERGe) for ADARs.

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

Methods for purification and characterization of nicked tRNAs.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.004

Bruno Costa, Valentina Blanco, Alfonso Cayota, Juan Pablo Tosar

引用次数: 0

Ribozyme-mediated expression of tRNA-derived small RNAs in bacteria.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.003

Carmela Esposito, Anamaria Buzoianu, Marina Cristodero, Norbert Polacek

{"title":"Ribozyme-mediated expression of tRNA-derived small RNAs in bacteria.","authors":"Carmela Esposito, Anamaria Buzoianu, Marina Cristodero, Norbert Polacek","doi":"10.1016/bs.mie.2024.11.003","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.003","url":null,"abstract":"Transfer RNA-derived RNAs (tDRs) have emerged as important regulatory molecules found across all three domains of life. Despite their discovery over four decades ago, their biological significance has only recently begun to be elucidated. However, studying bacterial tDRs poses challenges due to technical limitations in assessing their in vivo functionality. To address this, we established a novel approach utilizing a self-cleaving Twister ribozyme to express tDRs in Escherichia coli. Specifically, we employed the type P1 Sva1-1 Twister ribozyme, to generate tDRs with genuine 3' ends. Our method involves the inducible expression of tDRs by incorporating the desired tDR sequence into a plasmid construct downstream of two lac operators and upstream of the Twister ribozyme. Upon induction with IPTG and transcription of the construct, the Twister ribozyme undergoes self-cleavage, thus producing tDRs with defined 3' ends. As a proof of principle, we demonstrated the in vivo application of our novel method by expressing and analyzing two stress-induced tRNA halves in E. coli. Overall, our method offers a valuable tool for studying tDRs in bacteria to shed light on their regulatory roles in cellular processes.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"65-83"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425825","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.

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

TGIRT-seq to profile tRNA-derived RNAs and associated RNA modifications.

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-11-22 DOI: 10.1016/bs.mie.2024.11.001

Abigail Grace Johnston, Monima Anam, Anindya Dutta, Zhangli Su

{"title":"TGIRT-seq to profile tRNA-derived RNAs and associated RNA modifications.","authors":"Abigail Grace Johnston, Monima Anam, Anindya Dutta, Zhangli Su","doi":"10.1016/bs.mie.2024.11.001","DOIUrl":"10.1016/bs.mie.2024.11.001","url":null,"abstract":"RNA modifications are key regulators for RNA processes. tRNA-derived RNAs are small RNAs with size between 15 and 50 bases long that are processed from mature or precursor tRNAs. Despite their more recent discovery, tRNA-derived RNAs have been found to play regulatory roles in many cellular processes including gene silencing, protein synthesis, stress response, and transgenerational inheritance. Furthermore, tRNA-derived RNAs are highly abundant in bodily fluids, posing as potential biomarkers. A unique feature of tRNA-derived RNAs is that they are rich in RNA modifications. Many of the RNA modifications on tRNA-derived RNAs disrupt Watson-Crick base pairing and will thus stall reverse transcriptase, such as N1-methyladenosine (m1A), N1-methylguanosine (m1G) and N2, N2-dimethylguanosine (m22G). These RNA modifications add another layer of regulation onto tRNA-derived RNAs' functions and are of interests for future research. However, these RNA modifications could also lead to lower detection of modification-containing RNAs in genome-wide small RNA sequencing analysis due to reverse transcriptase stall. To circumvent this bias, TGIRT (Thermostable Group II Intron Reverse Transcriptase) has been used to readthrough RNA modifications inserting mismatches. These mismatch signatures can then be used to precisely map the modification sites at base resolution. Here we describe the step-by-step experimental protocol to start with purified RNAs from cells or tissues and use TGIRT to make small RNA sequencing library for Illumina sequencing to profile the abundance of tRNA-derived RNAs and the associated RNA modifications.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"223-240"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11890191/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425832","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