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Methods in enzymology最新文献

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Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-07 DOI: 10.1016/bs.mie.2025.01.011
Alberto Marin-Gonzalez, Adam T Rybczynski, Roger S Zou, Taekjip Ha
{"title":"Measuring double-strand break repair events in mammalian cells with multi-target CRISPR.","authors":"Alberto Marin-Gonzalez, Adam T Rybczynski, Roger S Zou, Taekjip Ha","doi":"10.1016/bs.mie.2025.01.011","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.011","url":null,"abstract":"<p><p>A mechanistic understanding of the different pathways involved in the repair of DSBs is a timely, yet challenging task. CRISPR-Cas9 is a powerful tool to induce DNA double-strand breaks (DSB) at defined genomic locations to study the ensuing repair response, but Cas9 studies are typically limited by i) low-throughput induction of DSB, by targeting only one or a few genomic sites, or ii) the use of genetically integrated reporter systems, which do not always reflect endogenous phenotypes. To address these limitations, we developed multi-target CRISPR, a Cas9-based tool to controllably induce DSBs in high-throughput at endogenous sites, by leveraging repetitive genomic regions. In this Chapter, we describe how to design and execute a multi-target CRISPR experiment. We also detail how to analyze next-generation sequencing data for characterization of DSB repair events at multiple cut sites. We envision that multi-target CRISPR will become a valuable tool for the study of mammalian DSB repair mechanisms.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"1-22"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692807","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 Cas13a expression and purification for use in CRISPR diagnostics.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-12 DOI: 10.1016/bs.mie.2025.01.030
Her Xiang Chai, Rebecca S Bamert, Gavin J Knott
{"title":"Methods for Cas13a expression and purification for use in CRISPR diagnostics.","authors":"Her Xiang Chai, Rebecca S Bamert, Gavin J Knott","doi":"10.1016/bs.mie.2025.01.030","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.030","url":null,"abstract":"<p><p>The threat of emerging infectious diseases (e.g., SARS-CoV-2 the RNA virus responsible for the COVID-19 pandemic) has highlighted the importance of accurate and rapid testing for screening, patient diagnosis, and effective treatment of infectious disease. Nucleic acid diagnostic tools such as qPCR are considered the gold standard, providing a sensitive, accurate, and robust method of detection. However, these conventional diagnostic platforms are resource intensive, limited in some applications, and are almost always confined to laboratory settings. With the increasing demand for low-cost, rapid, and accurate point-of-care diagnostics, CRISPR-based systems have emerged as powerful tools to augment detection capabilities. Of note is the potent RNA detection enzyme, Leptotrichia buccalis (Lbu) Cas13a, which is capable of rapid RNA detection in complex mixtures with or without pre-amplification. To support its wide-spread use, we describe a detailed method for the expression, purification, and validation of LbuCas13a for use in molecular diagnostics.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"225-244"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692822","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
Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.004
Grace N Hibshman, David W Taylor
{"title":"Visualizing the conformational landscape of CRISPR-Cas9 through kinetics-informed structural studies.","authors":"Grace N Hibshman, David W Taylor","doi":"10.1016/bs.mie.2025.01.004","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.004","url":null,"abstract":"<p><p>CRISPR-Cas9 has transformed genome editing through its programmability and versatility. Its DNA cleavage activity involves dynamic conformational changes during gRNA binding, DNA recognition, R-loop formation, and endonuclease activation. Understanding these molecular transitions is critical for improving the specificity and efficiency of Cas9, but this remains challenging precisely due to these rapid structural rearrangements. Early structural studies provided foundational insights but were limited to static states under catalytically inactive conditions. Cryo-EM has since enabled visualization of the dynamic nature of active Cas9, by enriching for specific conformations. This chapter introduces a kinetics-informed cryo-EM approach to capture the stepwise activation of Cas9 in real time. With thorough kinetic analyses, such as stopped-flow measurements of R-loop formation, we describe how to identify optimal timepoints to visualize key conformational states with cryo-EM. Integration of kinetic and structural data enables precise mapping of the conformational landscape of Cas9 and other dynamic enzymes, advancing our understanding of their molecular mechanisms and providing a framework for engineering enhanced variants.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"41-53"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692837","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
EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/bs.mie.2024.11.029
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":"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":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11908505/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Mouse models for understanding physiological functions of ADARs.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-09 DOI: 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}
引用次数: 0
Preface.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00055-2
Peter A Beal
{"title":"Preface.","authors":"Peter A Beal","doi":"10.1016/S0076-6879(25)00055-2","DOIUrl":"https://doi.org/10.1016/S0076-6879(25)00055-2","url":null,"abstract":"","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"710 ","pages":"xvii-xix"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143052992","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
Northern blotting for human pre-tRNA and tRNA-derived RNAs.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.013
Yoshika Takenaka, Katsuki Aoyama, Yasutoshi Akiyama
{"title":"Northern blotting for human pre-tRNA and tRNA-derived RNAs.","authors":"Yoshika Takenaka, Katsuki Aoyama, Yasutoshi Akiyama","doi":"10.1016/bs.mie.2024.11.013","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.013","url":null,"abstract":"<p><p>Northern blotting (NB) is a classic method for visualizing the length as well as the amount of specific RNA using gel separation and hybridization probes. As transfer RNA-derived RNAs (tDRs) are generated from mature tRNAs or pre-tRNAs, the ratio of tDR to mature tRNA or pre-tRNA will be a useful information about the efficiency of tDR production. By designing NB probes which hybridize to a mature tRNA of interest, the blot can simultaneously visualize the amount of tDRs as well as mature tRNAs and pre-tRNAs originated from the same gene, which is a significant advantage of NB. In this chapter, we present a protocol for the detection of tDRs or pre-tRNAs by NB using denaturing polyacrylamide gel electrophoresis and Digoxigenin-dUTP-tailed oligo DNA probes. Through example experiments, we show that tDRs originating from the same mature tRNA can be differentiated based on their length. We also show that our method can be applied to the evaluation of pre-tRNA processing.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"15-27"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425743","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
tRNA-derived RNAs that form tetramolecular assemblies.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-11-22 DOI: 10.1016/bs.mie.2024.11.014
Prakash Kharel
{"title":"tRNA-derived RNAs that form tetramolecular assemblies.","authors":"Prakash Kharel","doi":"10.1016/bs.mie.2024.11.014","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.014","url":null,"abstract":"<p><p>Transfer RNA (tRNA)-derived small RNAs (tDRs) are emerging as a novel class of regulatory molecules with significant implications in gene expression and cellular processes. These tDRs are generated through precise cleavage of precursor or mature tRNAs and can function in a sequence dependent manner or structure dependent manner. Recent studies have uncovered a unique subset of tDRs that can form tetramolecular assemblies, adding a new layer of complexity to their functional repertoire. Tetramolecular tDRs exhibit remarkable stability and functional diversity, influencing processes such as translation regulation, stress response, and cellular signaling. The assembly of these tDRs into tetramers is facilitated by guanine-rich sequence motifs which promote intermolecular interactions essential for their structure and biological activity. Understanding the formation, structural dynamics, and functional roles of tetramolecular tDRs offers new insights into tDR-mediated gene regulation and the potential development of RNA-based therapeutic strategies. This article aims to discuss a set of biochemical, biophysical, and reporter assay-based techniques that can be used to characterize G-quadruplex structures formed by tDRs.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"47-63"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425835","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
Preparation of high-purity RNPs of CRISPR-based DNA base editors.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-27 DOI: 10.1016/bs.mie.2025.01.019
Mitchell J McAndrew, Madeleine B King, Audrone Lapinaite
{"title":"Preparation of high-purity RNPs of CRISPR-based DNA base editors.","authors":"Mitchell J McAndrew, Madeleine B King, Audrone Lapinaite","doi":"10.1016/bs.mie.2025.01.019","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.019","url":null,"abstract":"<p><p>Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"277-315"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692825","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
Biochemical reconstitution of a type I-B CRISPR-associated transposon.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-26 DOI: 10.1016/bs.mie.2025.01.042
Shukun Wang, Leifu Chang
{"title":"Biochemical reconstitution of a type I-B CRISPR-associated transposon.","authors":"Shukun Wang, Leifu Chang","doi":"10.1016/bs.mie.2025.01.042","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.042","url":null,"abstract":"<p><p>CRISPR-associated transposons (CASTs) are potential gene editing tools because of their RNA-guided DNA insertion activity. It is essential to understand the mechanisms underlying the transposition for the application of CASTs. Here, we provide protocols for the biochemical reconstitution of a type I-B CAST for RNA-guided transposition. The procedures may be applicable to other types of CASTs and facilitate the mechanism studies of various CASTs.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"55-79"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692754","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
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