{"title":"CellREADR: An ADAR-based RNA sensor-actuator device.","authors":"Xiaolu Yang, Kehali Woldemichael, Xiao Guo, Shengli Zhao, Yongjun Qian, Z Josh Huang","doi":"10.1016/bs.mie.2024.11.027","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.027","url":null,"abstract":"<p><p>RNAs are central mediators of genetic information flow and gene regulation that underlie diverse cell types and cell states across species. Thus, methods that can sense and respond to RNA profiles in living cells will have broad applications in biology and medicine. CellREADR - Cell access through RNA sensing by Endogenous ADAR (adenosine deaminase acting on RNA), is a programmable RNA sensor-actuator technology that couples the detection of a cell-defining RNA to the translation of an effector protein to monitor and manipulate the cell. The CellREADR RNA device consists of a 5' sensor region complementary to a cellular RNA and a 3' protein payload coding region. Payload translation is gated by the removal of a STOP codon in the sensor region upon base pairing with the cognate cellular RNA through an ADAR-mediated A-to-I editing mechanism ubiquitous to metazoan cells. CellREADR thus represents a new generation of programmable RNA device for monitoring and manipulating animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed procedure for implementing CellREADR experiments in cell culture systems and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo protocol focuses on AAV-based delivery of CellREADR through expression vectors using brain tissue as an example. We describe current best practices and various experimental controls.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"207-227"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143053003","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2025-01-02DOI: 10.1016/bs.mie.2024.11.033
Emma Lamb, Dyuti Pant, Boyoon Yang, Heather A Hundley
{"title":"A probe-based capture enrichment method for detection of A-to-I editing in low abundance transcripts.","authors":"Emma Lamb, Dyuti Pant, Boyoon Yang, Heather A Hundley","doi":"10.1016/bs.mie.2024.11.033","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.033","url":null,"abstract":"<p><p>Exactly two decades ago, the ability to use high-throughput RNA sequencing technology to identify sites of editing by ADARs was employed for the first time. Since that time, RNA sequencing has become a standard tool for researchers studying RNA biology and led to the discovery of RNA editing sites present in a multitude of organisms, across tissue types, and in disease. However, transcriptome-wide sequencing is not without limitations. Most notably, RNA sequencing depth of a given transcript is correlated with expression, and sequencing depth impacts the ability to robustly detect RNA editing events. This chapter focuses on a method for enrichment of low-abundance transcripts that can facilitate more efficient sequencing and detection of RNA editing events. An important note is that while we describe aspects of the protocol important for capturing intron-containing transcripts, this probe-based enrichment method could be easily modified to assess editing within any low-abundance transcript. We also provide some perspectives on the current limitations as well as important future directions for expanding this technology to gain more insights into how RNA editing can impact transcript diversity.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"55-75"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052974","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2025-01-02DOI: 10.1016/bs.mie.2024.11.032
Alla Fishman, Ayelet T Lamm
{"title":"Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.","authors":"Alla Fishman, Ayelet T Lamm","doi":"10.1016/bs.mie.2024.11.032","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.032","url":null,"abstract":"<p><p>Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"285-302"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052991","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2024-12-04DOI: 10.1016/bs.mie.2024.11.028
Jia Wei Joel Heng, Meng How Tan
{"title":"Nanopore sequencing to detect A-to-I editing sites.","authors":"Jia Wei Joel Heng, Meng How Tan","doi":"10.1016/bs.mie.2024.11.028","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.028","url":null,"abstract":"<p><p>Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"187-205"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052990","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2024-12-05DOI: 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":"<p><p>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.</p>","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2024-12-04DOI: 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":"<p><p>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\".</p>","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2024-12-04DOI: 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":"https://doi.org/10.1016/bs.mie.2024.11.022","url":null,"abstract":"<p><p>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.</p>","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2025-01-02DOI: 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":"https://doi.org/10.1016/bs.mie.2024.11.030","url":null,"abstract":"<p><p>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.</p>","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2024-12-04DOI: 10.1016/bs.mie.2024.11.029
Alexandria L Quillin, Benoît Arnould, Steve D Knutson, Tatiana F Flores, Jennifer M Heemstra
{"title":"EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts.","authors":"Alexandria L Quillin, Benoît Arnould, Steve D Knutson, Tatiana F Flores, Jennifer M Heemstra","doi":"10.1016/bs.mie.2024.11.029","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.029","url":null,"abstract":"<p><p>Adenosine-to-inosine (A-to-I) editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a prevalent post-transcriptional modification that is vital for numerous biological functions. Given that this modification impacts global gene expression, RNA localization, and innate cellular immunity, dysregulation of A-to-I editing has unsurprisingly been linked to a variety of cancers and other diseases. However, our current understanding of the underpinning mechanisms that connect dysregulated A-to-I editing and disease processes remains limited. Widely used methods require RNA extraction and pooling that ultimately erases subcellular localization and cell-to-cell variation, which may be critical to understanding misregulation. To overcome these challenges, we recently developed Endonuclease V Immunostaining Assay (EndoVIA) to selectively detect and visualize A-to-I edited RNA in situ. In this chapter, we describe in detail how to prepare cell samples, stain A-to-I edited transcripts with EndoVIA, quantify global inosine abundance, and visualize the subcellular localization of inosine-containing RNAs at the single molecule level.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"99-130"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052987","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}
Methods in enzymologyPub Date : 2025-01-01Epub Date: 2025-01-09DOI: 10.1016/bs.mie.2024.11.024
Qinyi Zhang, Carl R Walkley
{"title":"Mouse models for understanding physiological functions of ADARs.","authors":"Qinyi Zhang, Carl R Walkley","doi":"10.1016/bs.mie.2024.11.024","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.024","url":null,"abstract":"<p><p>Adenosine-to-inosine (A-to-I) editing, is a highly prevalent posttranscriptional modification of RNA, mediated by the adenosine deaminases acting on RNA (ADAR) proteins. Mammalian transcriptomes contain tens of thousands to millions of A-to-I editing events. Mutations in ADAR can result in rare autoinflammatory disorders such as Aicardi-Goutières syndrome (AGS) through to irreversible conditions such as motor neuron disease, amyotrophic lateral sclerosis (ALS). Mouse models have played an important role in our current understanding of the physiology of ADAR proteins. With the advancement of genetic engineering technologies, a number of new mouse models have been recently generated, each providing additional insight into ADAR function. This review highlights both past and current mouse models, exploring the methodologies used in their generation, their respective discoveries, and the significance of these findings in relation to human ADAR physiology.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"153-185"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052989","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}
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