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Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins.
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
{"title":"Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins.","authors":"Andrew A Williams, Olivine Redman, Hannah Domgaard, Matthew J Armbrust, Ryan N Jackson","doi":"10.1016/bs.mie.2025.01.039","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.039","url":null,"abstract":"<p><p>Type IV CRISPR systems are phylogenetically diverse and poorly understood. However, recently, major strides have been made toward understanding type IV-A systems. In type IV-A systems, a multi-subunit ribonucleoprotein complex, called the Csf complex, uses a CRISPR-derived guide to bind double-stranded DNA, forming an R-loop to which a helicase called CRISPR-associated DinG (CasDinG) is recruited. It is proposed that the ATP-dependent helicase activity of CasDinG then unwinds duplex DNA near the targeting site, impairing RNA transcription, and gene expression. Here we describe methods used to investigate the type IV-A system from Pseudomonas aeruginosa strain 83 including a plasmid clearance assay, expression and purification of type IV ribonucleoprotein complexes and proteins, nucleic acid binding assays, and CasDinG helicase assays. These methods provide a foundation for future work aimed at understanding these enigmatic systems.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"79-114"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692767","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 Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-06 DOI: 10.1016/bs.mie.2025.01.006
Samuel I Gould, Francisco J Sánchez-Rivera
{"title":"Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.","authors":"Samuel I Gould, Francisco J Sánchez-Rivera","doi":"10.1016/bs.mie.2025.01.006","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.006","url":null,"abstract":"<p><p>Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"437-451"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692835","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.
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":"<p><p>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.</p>","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
Purification and in vivo, cell-free, and in vitro characterization of CRISPR-Cas12a2.
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":"<p><p>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.</p>","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
Functional characterization of tRNA-derived small RNAs in stem cells.
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":"https://doi.org/10.1016/bs.mie.2024.11.015","url":null,"abstract":"<p><p>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.</p>","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
In vitro functional analysis of plant tDRs.
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
{"title":"In vitro functional analysis of plant tDRs.","authors":"Christina Berrissou, Laurence Drouard","doi":"10.1016/bs.mie.2024.11.011","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.011","url":null,"abstract":"<p><p>In the world of small non-coding RNAs, tRNA-derived RNAs (tDRs) have emerged in recent years as being involved in a wide range of biological functions in every domain of life. In plants, our knowledge of the roles of tDRs is still very sparse. Nevertheless, the data produced to date demonstrate their importance in regulating gene expression at the transcriptional and post-transcriptional levels, during development, or in response to biotic and abiotic stresses. Studying the functions of plant tDRs in vivo is not an easy task, and in vitro studies offer an interesting alternative. Here we describe two in vitro approaches aimed at deciphering molecular mechanisms involving plant tDRs. On the one hand, we describe how to identify tDRs capable of inhibiting protein synthesis in vitro, and on the other, we explain how to use protoplast transfection to study the localization of tDRs and determine their protein interactome.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"203-221"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425727","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.
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
{"title":"Prime editing in bacteria with BacPE.","authors":"Hongyuan Zhang, Quanjiang Ji","doi":"10.1016/bs.mie.2025.01.026","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.026","url":null,"abstract":"<p><p>Programmable genome editing technologies have revolutionized the ability of researchers to alter the genomes of microorganisms in a straightforward and efficient manner, significantly advancing the field of microbiology. To date, several CRISPR-Cas-based genome-editing systems have been developed for use in E. coli, including CRISPR/Cas9, base editing, and prime editing technologies. In this chapter, we describe the design and experimental application of BacPE, a variant of prime editing technology optimized for E. coli. BacPE facilitates the introduction of point mutations, insertions, and deletions without the need for double-strand DNA breaks. We demonstrate that BacPE is a powerful tool for genome editing in E. coli and highlight its potential applicability to other bacterial species.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"405-418"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692743","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 general framework to analyze potential roles of tDRs in mammalian protein synthesis.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2024-12-05 DOI: 10.1016/bs.mie.2024.11.018
Nupur Bhatter, Pavel Ivanov
{"title":"A general framework to analyze potential roles of tDRs in mammalian protein synthesis.","authors":"Nupur Bhatter, Pavel Ivanov","doi":"10.1016/bs.mie.2024.11.018","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.018","url":null,"abstract":"<p><p>tRNA-derived RNAs (tDRs) are a heterogeneous class of small non-coding RNAs that have been implicated in numerous biological processes including the regulation of mRNA translation. A subclass of tDRs called tRNA-derived stress-induced RNAs (tiRNAs) have been shown to participate in translational control under stress where specific tiRNAs repress protein synthesis. Here, we use a prototypical tiRNA (5'-tiRNA<sup>Ala</sup>) that inhibits mRNA translation in vitro and in cells as a model to study potential roles of tDRs in translational control. Specifically, we propose to use commercially available and custom-made in vitro translation systems together with sensitive luciferase-based mRNA reporters as well as transfection studies to determine potential effects of a given tDR on various aspects of protein synthesis. We overview methods to probe the capacity of specific tDRs to target specific steps of mRNA translation initiation, the most regulated step in translational control. Using 5'-tiRNA<sup>Ala</sup> as an example, we analyze its effects on the integrity of the m<sup>7</sup>GTP (cap)-bound eIF4F complex and phosphorylation of eIF2α, the key regulatory molecule of the Integrated Stress Response. Using transfection studies, we also monitor whether tDRs can promote formation of stress granules (SGs), RNA granules are often formed in response to global translation repression in live cells. This simple workflow offers fast, scalable, and reliable analyses of a potential involvement of specific tDRs in the modulation of protein synthesis and provides initial hints on molecular mechanisms that underline such mRNA translation regulation.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"29-46"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425700","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
Cloning and validating systems for high throughput molecular recording.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-09 DOI: 10.1016/bs.mie.2025.01.015
Anqi Zhao, Michelle M Chan
{"title":"Cloning and validating systems for high throughput molecular recording.","authors":"Anqi Zhao, Michelle M Chan","doi":"10.1016/bs.mie.2025.01.015","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.015","url":null,"abstract":"<p><p>Molecular recording technologies record and store information about cellular history. Lineage tracing is one form of molecular recording and produces information describing cellular trajectories during mammalian development, differentiation and maintenance of adult stem cell niches, and tumor evolution. Our molecular recorder technology utilizes CRISPR-Cas9 barcode editing to generate mutations in genomically integrated, engineered DNA cassettes, which are read out by single-cell RNA sequencing and used to produce high-resolution lineage trees. Here, we describe optimized cloning and validation procedures to construct the molecular recorder lineage tracing system. We include information on considerations of technology design, cloning procedures, the generation of lineage tracing cell lines, and time course experiments to assess their performance.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"453-473"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692757","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
Delivery of genome editors with engineered virus-like particles.
4区 生物学
Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-09 DOI: 10.1016/bs.mie.2025.01.007
Christopher Lu, Yuanhang Li, Jacob Ryan Cummings, Samagya Banskota
{"title":"Delivery of genome editors with engineered virus-like particles.","authors":"Christopher Lu, Yuanhang Li, Jacob Ryan Cummings, Samagya Banskota","doi":"10.1016/bs.mie.2025.01.007","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.007","url":null,"abstract":"<p><p>Genome editing technologies have revolutionized biomedical sciences and biotechnology. However, their delivery in vivo remains one of the major obstacles for clinical translation. Here, we introduce various emerging genome editing systems and review different delivery systems have been developed to realize the promise of in vivo gene editing therapies. In particular, we focus on virus-like particles (VLPs), an emerging delivery platform and provide in depth analysis on recent advancements to improve VLPs delivery potential and highlight opportunities for future improvements. To this end, we also provide detail workflows for engineered VLP (eVLP) selection, production, and purification, along with methods for characterization and validation.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"712 ","pages":"475-516"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143692763","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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