发布求助
文献互助 智能选刊 最新文献

Methods in enzymology最新文献

筛选
英文 中文
Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries. 利用质数编辑引导生成器（peg）实现高通量生成质数编辑传感器库。
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
Methods for purification and characterization of nicked tRNAs. nick trna的纯化和表征方法。
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
{"title":"Methods for purification and characterization of nicked tRNAs.","authors":"Bruno Costa, Valentina Blanco, Alfonso Cayota, Juan Pablo Tosar","doi":"10.1016/bs.mie.2024.11.004","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.004","url":null,"abstract":"<p><p>While tRNA-derived fragments (tDRs) play important roles in gene expression regulation, it is technically challenging to distinguish bona fide tDRs from nicked tRNAs. This is because analytical techniques used to study RNA, such as northern blot, RT-qPCR or sequencing involve the use of denaturing reagents (e.g., phenol, formamide, urea) or physical procedures (e.g., heat) that convert nicked tRNAs into tRNA halves or other tDRs. In this chapter, we describe a protocol that enables the purification of nicked tRNAs under non-denaturing conditions that preserve their 3D structure. Purified nicked tRNAs can then be either enzymatically repaired into almost full-length tRNAs, or chromatographically separated from single-stranded tDRs before detection. These protocols will allow researchers to distinguish between structurally distinct but sequence identical tDRs and nicked tRNAs, disentangling their biological functions.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"711 ","pages":"187-201"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425733","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
Ribozyme-mediated expression of tRNA-derived small RNAs in bacteria. 核糖酶介导的trna衍生小rna在细菌中的表达。
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":"<p><p>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.</p>","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
TGIRT-seq to profile tRNA-derived RNAs and associated RNA modifications. TGIRT-seq分析trna衍生RNA和相关RNA修饰。
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":"<p><p>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 N<sup>1</sup>-methyladenosine (m<sup>1</sup>A), N<sup>1</sup>-methylguanosine (m<sup>1</sup>G) and N<sup>2</sup>, N<sup>2</sup>-dimethylguanosine (m<sup>2</sup><sub>2</sub>G). 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.</p>","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
Functional characterization of tRNA-derived small RNAs in stem cells. 干细胞中trna衍生小rna的功能表征。
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. 植物tDRs的离体功能分析。
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. 带BacPE的细菌启动编辑。
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
Cloning and validating systems for high throughput molecular recording. 克隆和验证系统的高通量分子记录。
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. 基因编辑器与工程病毒样颗粒的传递。
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
Measuring double-strand break repair events in mammalian cells with multi-target CRISPR. 用多靶点CRISPR测量哺乳动物细胞中的双链断裂修复事件。
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
首页 上一页 下一页 尾页
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整，为了账户安全，请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
请完成安全验证×
相关产品
×
本文献相关产品
联系我们：info@booksci.cn Book学术提供免费学术资源搜索服务，方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号：481959085
Book学术官方微信