209 results on '"Rna targeting"'
Search Results
2. A kind of magic
- Author
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Hansford, Dave
- Published
- 2024
3. Design, Synthesis, and Evaluation of Neomycin‐Imidazole Conjugates for RNA Cleavage.
- Author
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Martin, Céline, Bonnet, Maurinne, Patino, Nadia, Azoulay, Stéphane, Di Giorgio, Audrey, and Duca, Maria
- Subjects
- *
BINDING sites , *RNA , *SMALL molecules , *NON-coding RNA , *MOLECULAR docking - Abstract
Targeting RNA with synthetic small molecules attracted much interest during recent years as a particularly promising therapeutic approach in a large number of pathologies spanning from genetic disorders, cancers as well as bacterial and viral infections. In this work, we took advantage of a known RNA binder, neomycin, to prepare neomycin‐imidazole conjugates mimicking the active site of ribonuclease enzymes able to induce a site‐specific cleavage of HIV‐1 TAR RNA in physiological conditions. These new conjugates were prepared using a straightforward synthetic methodology and were studied for their ability to bind the target, inhibit Tat/TAR interaction and induce selective cleavage using fluorescence‐based assays and molecular docking. We found compounds with nanomolar affinity, promising cleavage activity and the ability to inhibit Tat/TAR interaction with submicromolar IC50s. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
4. RNA and Life Threatening Diseases
- Author
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Rami, Esha and Rami, Esha
- Subjects
- Medical genetics, Genetic regulation, RNA targeting
- Abstract
A huge number of messenger RNAs (mRNAs) and non-coding RNAs must be accurately expressed for cells to function normally. These RNAs take a role in transcription, RNA processing, and translation. An in-depth examination of RNA-mediated genome regulation at several levels has been provided in the book RNA-based Regulation in Human Health and Disease. Starting with the Introduction of RNA, RNA as a therapeutic target, a further section examines the various diseases and significant potential for RNA-based medicines and diagnostics in the future. The book helps researchers, students and clinicians across the world who will find this book very informative as well as practical.
- Published
- 2023
5. Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d
- Author
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Zhang, Cheng, Konermann, Silvana, Brideau, Nicholas J, Lotfy, Peter, Wu, Xuebing, Novick, Scott J, Strutzenberg, Timothy, Griffin, Patrick R, Hsu, Patrick D, and Lyumkis, Dmitry
- Subjects
Genetics ,Bioengineering ,Biotechnology ,Underpinning research ,1.1 Normal biological development and functioning ,Quality Education ,CRISPR-Cas Systems ,Clustered Regularly Interspaced Short Palindromic Repeats ,Cryoelectron Microscopy ,Endonucleases ,HEK293 Cells ,Humans ,Molecular Conformation ,RNA ,RNA ,Guide ,Kinetoplastida ,Ribonucleases ,CRISPR ,Cas13 ,Cas13d ,Cas9 ,RNA interference ,RNA targeting ,cryo-EM ,shRNA ,structure ,type VI ,Biological Sciences ,Medical and Health Sciences ,Developmental Biology - Abstract
CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
- Published
- 2018
6. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors
- Author
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Konermann, Silvana, Lotfy, Peter, Brideau, Nicholas J, Oki, Jennifer, Shokhirev, Maxim N, and Hsu, Patrick D
- Subjects
Acquired Cognitive Impairment ,Dental/Oral and Craniofacial Disease ,Biotechnology ,Alzheimer's Disease including Alzheimer's Disease Related Dementias (AD/ADRD) ,Human Genome ,Brain Disorders ,Neurosciences ,Dementia ,Genetics ,Quality Education ,Alternative Splicing ,Animals ,Bacterial Proteins ,CRISPR-Cas Systems ,Cell Differentiation ,Computational Biology ,Escherichia coli ,Gene Expression Profiling ,Genetic Engineering ,HEK293 Cells ,Humans ,Induced Pluripotent Stem Cells ,Lentivirus ,Mice ,Protein Engineering ,RNA ,RNA Interference ,RNA ,Guide ,Kinetoplastida ,Ruminococcus ,Sequence Analysis ,RNA ,Transcriptome ,CRISPR ,Cas13 ,CasRx ,RNA interference ,RNA targeting ,alternative splicing ,frontotemporal dementia ,gene editing ,genome engineering ,tau ,Biological Sciences ,Medical and Health Sciences ,Developmental Biology - Abstract
Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.
- Published
- 2018
7. Applications of CRISPR/Cas13-Based RNA Editing in Plants.
- Author
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Kavuri, Naga Rajitha, Ramasamy, Manikandan, Qi, Yiping, and Mandadi, Kranthi
- Subjects
- *
PLANT RNA , *RNA editing , *GENOME editing , *CRISPRS , *PLANT resistance to viruses - Abstract
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
8. Engineered U1 snRNAs to modulate alternatively spliced exons.
- Author
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Hatch, Samuel T., Smargon, Aaron A., and Yeo, Gene W.
- Subjects
- *
SMALL nuclear RNA , *ALTERNATIVE RNA splicing , *SPINAL muscular atrophy , *DUCHENNE muscular dystrophy , *DYSAUTONOMIA , *GENOME editing - Abstract
• Engineering of sequence-specific alternative splicing modulators. • Robust exclusion of endogenous FAS exon 6 and inclusion of endogenous SMN2 exon 7. • Underscoring U1 snRNA as a candidate therapeutic and research tool. Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool. With the goal of providing a user-friendly protocol, we compile and demonstrate a methodological toolkit for sequence-specific targeted perturbation of alternatively spliced pre-mRNA with engineered U1 snRNAs. We observe robust modulation of endogenous pre-mRNA transcripts targeted in two contrasting splicing contexts, SMN2 exon 7 and FAS exon 6, exhibiting the utility and applicability of engineered U1 snRNA to both inclusion and exclusion of targeted exons. We anticipate that these demonstrations will contribute to the usability of U1 snRNA in investigating splicing modulation in eukaryotic cells, increasing accessibility to the broader research community. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
9. Mechanisms and applications of peptide nucleic acids selectively binding to double‐stranded RNA.
- Author
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Zhan, Xuan, Deng, Liping, and Chen, Gang
- Abstract
RNAs form secondary structures containing double‐stranded base paired regions and single‐stranded regions. Probing, detecting and modulating RNA structures and dynamics requires the development of molecular sensors that can differentiate the sequence and structure of RNAs present in viruses and cells, as well as in extracellular space. In this review, we summarize the recent progress on the development of chemically modified peptide nucleic acids (PNAs) for the selective recognition of double‐stranded RNA (dsRNA) sequences over both single‐stranded RNA (ssRNA) and double‐stranded DNA (dsDNA) sequences. We also briefly discuss the applications of sequence‐specific dsRNA‐binding PNAs in sensing and stabilizing dsRNA structures and inhibiting dsRNA‐protein interactions. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
10. Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1
- Author
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Raul Ondono, Ángel Lirio, Carlos Elvira, Elena Álvarez-Marimon, Claudia Provenzano, Beatrice Cardinali, Manuel Pérez-Alonso, Alex Perálvarez-Marín, José I. Borrell, Germana Falcone, and Roger Estrada-Tejedor
- Subjects
Myotonic dystrophy ,Molecular modelling ,RNA targeting ,Small molecule ,Base recognition ,Biotechnology ,TP248.13-248.65 - Abstract
Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3′ untranslated region (3′UTR). The intrinsic complexity and lack of crystallographic data makes noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequester many essential alternative splicing factors such as Muscleblind-like 1 (MBNL1). Most DM1 phenotypic features stem from the reduced availability of free MBNL1 and therefore many therapeutic efforts are focused on recovering its normal activity. For that purpose, herein we present pyrido[2,3-d]pyrimidin-7-(8H)-ones, a privileged scaffold showing remarkable biological activity against many targets involved in human disorders including cancer and viral diseases. Their combination with a flexible linker meets the requirements to stabilise DM1 toxic transcripts, and therefore, enabling the release of MBNL1. Therefore, a set of novel pyrido[2,3-d]pyrimidin-7-(8H)-ones derivatives (1a-e) were obtained using click chemistry. 1a exerted over 20% MBNL1 recovery on DM1 toxic RNA activity in primary cell biology studies using patient-derived myoblasts. 1a promising anti DM1 activity may lead to subsequent generations of ligands, highlighting a new affordable treatment against DM1.
- Published
- 2021
- Full Text
- View/download PDF
11. Progress of CRISPR-Cas13 Mediated Live-Cell RNA Imaging and Detection of RNA-Protein Interactions
- Author
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Huake Cao, Yuechen Wang, Ning Zhang, Siyuan Xia, Pengfei Tian, Li Lu, Juan Du, and Yinan Du
- Subjects
CRISPR ,Cas13 ,RNA imaging ,RNA-protein interactions ,RNA targeting ,RNA biology ,Biology (General) ,QH301-705.5 - Abstract
Ribonucleic acid (RNA) and proteins play critical roles in gene expression and regulation. The relevant study increases the understanding of various life processes and contributes to the diagnosis and treatment of different diseases. RNA imaging and mapping RNA-protein interactions expand the understanding of RNA biology. However, the existing methods have some limitations. Recently, precise RNA targeting of CRISPR-Cas13 in cells has been reported, which is considered a new promising platform for RNA imaging in living cells and recognition of RNA-protein interactions. In this review, we first described the current findings on Cas13. Furthermore, we introduced current tools of RNA real-time imaging and mapping RNA-protein interactions and highlighted the latest advances in Cas13-mediated tools. Finally, we discussed the advantages and disadvantages of Cas13-based methods, providing a set of new ideas for the optimization of Cas13-mediated methods.
- Published
- 2022
- Full Text
- View/download PDF
12. Controlling Site‐Directed RNA Editing by Chemically Induced Dimerization.
- Author
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Stroppel, Anna S., Lappalainen, Ruth, and Stafforst, Thorsten
- Subjects
- *
RNA editing , *DIMERIZATION , *GIBBERELLIC acid , *STAT proteins , *CELL culture - Abstract
Various RNA‐targeting approaches have been engineered to modify specific sites on endogenous transcripts, breaking new ground for a variety of basic research tools and promising clinical applications in the future. Here, we combine site‐directed adenosine‐to‐inosine RNA editing with chemically induced dimerization. Specifically, we achieve tight and dose‐dependent control of the editing reaction with gibberellic acid, and obtain editing yields up to 20 % and 44 % in the endogenous STAT1 and GAPDH transcript in cell culture. Furthermore, the disease‐relevant MECP2 R106Q mutation was repaired with editing yields up to 42 %. The introduced principle will enable new applications where temporal or spatiotemporal control of an RNA‐targeting mechanism is desired. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
13. Applications of CRISPR/Cas13-Based RNA Editing in Plants
- Author
-
Naga Rajitha Kavuri, Manikandan Ramasamy, Yiping Qi, and Kranthi Mandadi
- Subjects
type VI CRISPR systems ,CRISPR/Cas13 ,RNA targeting ,RNA editing ,RNA interference ,Cytology ,QH573-671 - Abstract
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants.
- Published
- 2022
- Full Text
- View/download PDF
14. A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells.
- Author
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Tieu, Victor, Sotillo, Elena, Bjelajac, Jeremy R., Chen, Crystal, Malipatlolla, Meena, Guerrero, Justin A., Xu, Peng, Quinn, Patrick J., Fisher, Chris, Klysz, Dorota, Mackall, Crystal L., and Qi, Lei S.
- Subjects
- *
T cells , *T-cell exhaustion , *METABOLIC regulation , *ERGONOMICS , *TRANSCRIPTOMES , *CELL physiology - Abstract
CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond. [Display omitted] • MEGA allows massively multiplexed RNA knockdown in primary human T cells • Combinatorial knockdown screen identifies paired regulators of CAR T cell function • Tunable and reversible multi-gene regulation with an FDA-approved drug • Metabolic engineering enhances CAR T cell fitness and anti-tumor activity in vivo MEGA is a programmable RNA-targeting platform that enables safe and effective multiplexed genetic perturbation of the primary human T cell transcriptome. Multi-gene disruption broadly enhances the anti-tumor activity of CAR T cells and uncovers a role for aerobic glycolysis in driving T cell exhaustion. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
15. A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1
- Author
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Nan Zhang, Brittani Bewick, Guangbin Xia, Denis Furling, and Tetsuo Ashizawa
- Subjects
myotonic dystrophy ,neurodegeneration ,CRISPR-Cas13a ,RNA targeting ,stress granule ,Genetics ,QH426-470 - Abstract
Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii (Lsh) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) – a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that LshCas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, LshCas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated LshCas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of LshCas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases.
- Published
- 2020
- Full Text
- View/download PDF
16. A CRISPR-Cas13a Based Strategy That Tracks and Degrades Toxic RNA in Myotonic Dystrophy Type 1.
- Author
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Zhang, Nan, Bewick, Brittani, Xia, Guangbin, Furling, Denis, and Ashizawa, Tetsuo
- Subjects
MYOTONIA atrophica ,RNA ,MYOBLASTS ,NEUROMUSCULAR diseases ,ORGANELLES ,MICROSATELLITE repeats - Abstract
Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii (Lsh) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) – a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that Lsh Cas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, Lsh Cas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated Lsh Cas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of Lsh Cas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
17. In Vitro Evaluation of Bis-3-Chloropiperidines as RNA Modulators Targeting TAR and TAR-Protein Interaction
- Author
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Alice Sosic, Giulia Olivato, Caterina Carraro, Richard Göttlich, Dan Fabris, and Barbara Gatto
- Subjects
RNA targeting ,RNA-based interactions ,bis-3-chloropiperidines ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
After a long limbo, RNA has gained its credibility as a druggable target, fully earning its deserved role in the next generation of pharmaceutical R&D. We have recently probed the trans-activation response (TAR) element, an RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabilizing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analytical approaches for the study of multicomponent RNA-based interactions.
- Published
- 2022
- Full Text
- View/download PDF
18. Photodynamic Therapy with Tumor Cell Discrimination through RNA-Targeting Ability of Photosensitizer
- Author
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Yuan Xu, Yang Tan, Xiuqin Ma, Xiaoyi Jin, Ye Tian, and Miao Li
- Subjects
photosensitizer ,cyanine dyes ,RNA targeting ,cancer cell discrimination ,Organic chemistry ,QD241-441 - Abstract
Photodynamic therapy (PDT) represents an effective treatment to cure cancer. The targeting ability of the photosensitizer is of utmost importance. Photosensitizers that discriminate cancer cells can avoid the killing of normal cells and improve PDT efficacy. However, the design and synthesis of photosensitizers conjugated with a recognition unit of cancer cell markers is complex and may not effectively target cancer. Considering that the total RNA content in cancer cells is commonly higher than in normal cells, this study has developed the photosensitizer QICY with RNA-targeting abilities for the discrimination of cancer cells. QICY was specifically located in cancer cells rather than normal cells due to their stronger electrostatic interactions with RNA, thereby further improving the PDT effects on the cancer cells. After intravenous injection into mice bearing a xenograft tumor, QICY accumulated into the tumor location through the enhanced permeability and retention effect, automatically targeted cancer cells under the control of RNA, and inhibited tumor growth under 630 nm laser irradiation without obvious side effects. This intelligent photosensitizer with RNA-targeting ability not only simplifies the design and synthesis of cancer-cell-targeting photosensitizers but also paves the way for the further development of highly efficient PDTs.
- Published
- 2021
- Full Text
- View/download PDF
19. Progress of CRISPR-based programmable RNA manipulation and detection.
- Author
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Wang B and Yang H
- Subjects
- CRISPR-Cas Systems, Ribonucleoproteins genetics, RNA genetics, Gene Editing methods
- Abstract
Prokaryotic clustered regularly interspaced short palindromic repeats and CRISPR associated (CRISPR-Cas) systems provide adaptive immunity by using RNA-guided endonucleases to recognize and eliminate invading foreign nucleic acids. Type II Cas9, type V Cas12, type VI Cas13, and type III Csm/Cmr complexes have been well characterized and developed as programmable platforms for selectively targeting and manipulating RNA molecules of interest in prokaryotic and eukaryotic cells. These Cas effectors exhibit remarkable diversity of ribonucleoprotein (RNP) composition, target recognition and cleavage mechanisms, and self discrimination mechanisms, which are leveraged for various RNA targeting applications. Here, we summarize the current understanding of mechanistic and functional characteristics of these Cas effectors, give an overview on RNA detection and manipulation toolbox established so far including knockdown, editing, imaging, modification, and mapping RNA-protein interactions, and discuss the future directions for CRISPR-based RNA targeting tools. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications., (© 2023 Wiley Periodicals LLC.)
- Published
- 2023
- Full Text
- View/download PDF
20. Generation of virus‐resistant potato plants by RNA genome targeting.
- Author
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Zhan, Xiaohui, Zhang, Fengjuan, Zhong, Ziyang, Chen, Ruhao, Wang, Yong, Chang, Ling, Bock, Ralph, Nie, Bihua, and Zhang, Jiang
- Subjects
- *
POTATOES , *PLANT viruses , *PLANT RNA - Abstract
Summary: CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA‐targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
21. Advances in CRISPR-Cas systems for RNA targeting, tracking and editing.
- Author
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Wang, Fei, Wang, Lianrong, Zou, Xuan, Duan, Suling, Li, Zhiqiang, Deng, Zixin, Luo, Jie, Lee, Sang Yup, and Chen, Shi
- Subjects
- *
RNA , *RNA editing , *SYSTEMS development , *AMINO acids - Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely used in gene/genome targeting. Modifications of Cas9 enable these systems to become platforms for precise DNA manipulations. However, the utilization of CRISPR-Cas systems in RNA targeting remains preliminary. The discovery of type VI CRISPR-Cas systems (Cas13) shed light on RNA-guided RNA targeting. Cas13d, the smallest Cas13 protein, with a length of only ~930 amino acids, is a promising platform for RNA targeting compatible with viral delivery systems. Much effort has also been made to develop Cas9, Cas13a and Cas13b applications for RNA-guided RNA targeting. The discovery of new RNA-targeting CRISPR-Cas systems as well as the development of RNA-targeting platforms with Cas9 and Cas13 will promote RNA-targeting technology substantially. Here, we review new advances in RNA-targeting CRISPR-Cas systems as well as advances in applications of these systems in RNA targeting, tracking and editing. We also compare these Cas protein-based technologies with traditional technologies for RNA targeting, tracking and editing. Finally, we discuss remaining questions and prospects for the future. • RNA targeting and editing are becoming increasingly important • CRISPR-Cas systems are advancing for RNA targeting, tracking and editing • The type VI CRISPR-Cas systems are useful for RNA-guided RNA targeting • Use of Cas9 and Cas13 will advance RNA-targeting technologies [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
22. Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA.
- Author
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Dai, Yumin, Peralta, Ashley N., Wynn, Jessica E., Sherpa, Chringma, Li, Hao, Verma, Astha, Le Grice, Stuart F.J., and Santos, Webster L.
- Subjects
- *
RNA , *MOLECULAR recognition , *BINDING sites , *LEAD compounds - Abstract
Graphical abstract Abstract Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K d value of 410 nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
23. RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences
- Author
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Merel Derksen, Vicky Mertens, and Ger J.M. Pruijn
- Subjects
external guide sequence ,RNase P ,RNA cleavage ,RNA knockdown ,RNA targeting ,Microbiology ,QR1-502 - Abstract
The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels.
- Published
- 2015
- Full Text
- View/download PDF
24. The effects of length and sequence of gRNA on Cas13b and Cas13d activity in vitro and in vivo.
- Author
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Liu Y, Jing P, Zhou Y, Zhang J, Shi J, Zhang M, Yang H, and Fei J
- Subjects
- Animals, Mice, RNA, Guide, CRISPR-Cas Systems, Mammals genetics, RNA, CRISPR-Cas Systems genetics
- Abstract
Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d. There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock-down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA., (© 2023 Wiley-VCH GmbH.)
- Published
- 2023
- Full Text
- View/download PDF
25. Research progress in mRNA drug modification and delivery systems.
- Author
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Zhou Z and Li X
- Subjects
- RNA, Messenger genetics, Cytoplasm, Precision Medicine, Nanoparticles
- Abstract
Messenger RNA (mRNA) has shown tremendous potential in disease prevention and therapy. The clinical application requires mRNA with enhanced stability and high translation efficiency, ensuring it not to be degraded by nucleases and targeting to specific tissues and cells. mRNA immunogenicity can be reduced by nucleotide modification, and translation efficiency can be enhanced by codon optimization. The 5´ capping structure and 3´ poly A increase mRNA stability, and the addition of 5' and 3' non-translational regions regulate mRNA translation initiation and protein production. Nanoparticle delivery system protects mRNA from degradation by ubiquitous nucleases, enhances mRNA concentration in circulation and assists it cytoplasmic entrance for the purpose of treatment and prevention. Here, we review the recent advances of mRNA technology, discuss the methods and principles to enhance mRNA stability and translation efficiency; summarize the requirements involved in designing mRNA delivery systems with the potential for industrial translation and biomedical application. Furthermore, we provide insights into future directions of mRNA therapeutics to meet the needs for personalized precision medicine.
- Published
- 2023
- Full Text
- View/download PDF
26. Investigating the NRAS 5′ UTR as a target for small molecules.
- Author
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Balaratnam, Sumirtha, Torrey, Zachary R., Calabrese, David R., Banco, Michael T., Yazdani, Kamyar, Liang, Xiao, Fullenkamp, Christopher R., Seshadri, Srinath, Holewinski, Ronald J., Andresson, Thorkell, Ferré-D'Amaré, Adrian R., Incarnato, Danny, and Schneekloth, John S.
- Subjects
- *
ACUTE myeloid leukemia , *QUADRUPLEX nucleic acids , *SMALL molecules , *ANTISENSE DNA , *GENETIC translation , *MESSENGER RNA - Abstract
Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. The 5′ untranslated region (UTR) (5′ UTR) of the NRAS mRNA contains a G-quadruplex (G4) that regulates translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5′ UTR of the NRAS mRNA. We used a small molecule microarray screen to identify molecules that selectively bind to the NRAS -G4 with submicromolar affinity. One compound inhibits the translation of NRAS in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends and RT-PCR analysis revealed that the predominant NRAS transcript does not possess the G4 structure. Thus, although NRAS transcripts lack a G4 in many cell lines the concept of targeting folded regions within 5′ UTRs to control translation remains a highly attractive strategy. [Display omitted] • SMMs enable identification of a lead compound which binds to a G4 in the NRAS mRNA • Chemical inhibition of translation by targeting G4 in the 5 ′ -UTR of the NRAS mRNA • Analysis indicates heterogeneity in in the length of the NRAS mRNA 5′-UTR Balaratnam et al. used a high throughput small molecule microarray screen to identify novel small molecules that block translation by binding to a G-quadruplex structure from the NRAS mRNA. Detailed chemical, biophysical, structural, and biological evaluation provide insights into both the structure and targetability of this cancer-associated mRNA, revealing transcript heterogeneity as a challenge to targeting NRAS translation. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
27. Locked Nucleic Acid: High-Affinity Targeting of Complementary RNA for RNomics
- Author
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Kauppinen, S., Vester, B., Wengel, J., Starke, K., editor, Born, G.V.R., editor, Eichelbaum, M., editor, Ganten, D., editor, Hofmann, F., editor, Rosenthal, W., editor, Rubanyi, G., editor, Erdmann, Volker, editor, Barciszewski, Jan, editor, and Brosius, Jürgen, editor
- Published
- 2006
- Full Text
- View/download PDF
28. Photoswitches for controllable RNA binding: a future approach in the RNA-targeting therapy
- Author
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Daria V. Berdnikova
- Subjects
Light ,Computer science ,Drug target ,Metals and Alloys ,HIV ,RNA ,Stereoisomerism ,General Chemistry ,Computational biology ,Ligands ,Benzylidene Compounds ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Materials Chemistry ,Ceramics and Composites ,Humans ,Spatiotemporal resolution ,Rna targeting ,Azo Compounds ,Binding selectivity - Abstract
RNA is an emerging drug target that opens new perspectives in the treatment of viral and bacterial infections, cancer and a range of so far incurable genetic diseases. Among the various strategies towards the design and development of selective and efficient ligands for targeting and detection of therapeutically relevant RNA, photoswitchable RNA binders represent a very promising approach due to the possibility to control the ligand-RNA and protein-RNA interactions by light with high spatiotemporal resolution. However, the field of photoswitchable RNA binders still remains underexplored due to challenging design of lead structures that should combine high RNA binding selectivity with efficient photochemical performance. The aim of this highlight article is to describe the development of photoswitchable noncovalent RNA binders and to outline the current situation and perspectives of this emerging interdisciplinary field.
- Published
- 2021
29. Corrigendum: Aminoglycoside Conjugation for RNA Targeting: Antimicrobials and Beyond
- Author
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Maria Duca, Audrey Di Giorgio, and Klara Aradi
- Subjects
Chemistry ,medicine.drug_class ,Organic Chemistry ,Aminoglycoside ,Antibiotics ,medicine ,RNA ,General Chemistry ,Antimicrobial ,Rna targeting ,Catalysis ,Microbiology - Published
- 2020
30. Development of Optimized Inhibitor RNAs Allowing Multisite-Targeting of the HCV Genome.
- Author
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Romero-López, Cristina, Lahlali, Thomas, Berzal-Herranz, Beatriz, and Berzal-Herranz, Alfredo
- Abstract
Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome’s internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
31. Synthesis, hybridization and fluorescence properties of a 2′-C-pyrene-triazole modified arabino-uridine nucleotide.
- Author
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Kumar, Pawan, Sharma, Pawan K., and Nielsen, Poul
- Subjects
- *
DRUG synthesis , *ARABINOSE , *URIDINE , *GENE targeting , *NUCLEIC acid hybridization - Abstract
A new pyrene-modified nucleotide monomer is introduced, wherein pyrene is attached to the 2′-position of arabino -uridine through a triazolemethyl linker. When the monomer is introduced into oligonucleotides, very stable DNA duplexes and three way junctions are obtained. An oligonucleotide featuring two modifications in the center shows four-fold increase in the intensity of the pyrene excimer signal on hybridization with an RNA target but not with a DNA target. The new nucleotide monomer has potential in DNA invader probes as well as in RNA targeting and detection. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
32. Aminoglycoside Conjugation for RNA Targeting: Antimicrobials and Beyond
- Author
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Maria Duca, Klara Aradi, and Audrey Di Giorgio
- Subjects
010405 organic chemistry ,Chemistry ,medicine.drug_class ,Organic Chemistry ,Antibiotics ,Aminoglycoside ,RNA ,Translation (biology) ,General Chemistry ,Ribosomal RNA ,Ligands ,010402 general chemistry ,Antimicrobial ,01 natural sciences ,Catalysis ,Anti-Bacterial Agents ,0104 chemical sciences ,RNA, Bacterial ,Aminoglycosides ,Biochemistry ,Protein biosynthesis ,medicine ,Rna targeting - Abstract
Natural aminoglycosides are therapeutically useful antibiotics and very efficient RNA ligands. They are oligosaccharides that contain several ammonium groups able to interfere with the translation process in prokaryotes upon binding to bacterial ribosomal RNA (rRNA), and thus, impairing protein synthesis. Even if aminoglycosides are commonly used in therapy, these RNA binders lack selectivity and are able to bind to a wide number of RNA sequences/structures. This is one of the reasons for their toxicity and limited applications in therapy. At the same time, the ability of aminoglycosides to bind to various RNAs renders them a great source of inspiration for the synthesis of new binders with improved affinity and specificity toward several therapeutically relevant RNA targets. Thus, a number of studies have been performed on these complex and highly functionalized compounds, leading to the development of various synthetic methodologies toward the synthesis of conjugated aminoglycosides. The aim of this review is to highlight recent progress in the field of aminoglycoside conjugation, paying particular attention to modifications performed toward the improvement of affinity and especially to the selectivity of the resulting compounds. This will help readers to understand how to introduce a desired chemical modification for future developments of RNA ligands as antibiotics, antiviral, and anticancer compounds.
- Published
- 2020
33. Clinical pharmacology of biological medicines
- Author
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Henry Fok and Tiago Soares
- Subjects
Clinical pharmacology ,biology ,medicine.drug_class ,business.industry ,General Medicine ,Bioinformatics ,Monoclonal antibody ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,law ,030220 oncology & carcinogenesis ,medicine ,biology.protein ,030212 general & internal medicine ,Antibody ,Rna targeting ,business ,Large size - Abstract
Biological medicines encompass a wide group of therapeutic agents that are manufactured by living organisms and include monoclonal antibodies, peptides (e.g. insulin), vaccines, blood products, RNA targeting therapies and gene and cellular therapies. Here, we focus on the clinical pharmacology of monoclonal antibodies, which occupy an increasing share of the market for a diverse set of indications. Their large size, complex structure and ability to bind to highly specific target sites, and the heterogeneity imparted by the nature of their production, mean that monoclonal antibodies exhibit unique and specific pharmacokinetic and pharmacodynamic characteristics. Therefore it has become increasingly important for all clinicians to become familiar with their properties and the ways in which they differ from traditional small chemical drugs. Although most monoclonal antibodies are prescribed under specialist supervision, most prescribers are likely to encounter patients given antibody therapies.
- Published
- 2020
34. RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering
- Author
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Yilan J. Shi, Aaron A. Smargon, and Gene W. Yeo
- Subjects
Scientific discovery ,Computational biology ,Article ,Animals, Genetically Modified ,Transcriptome ,DNA Endonuclease ,03 medical and health sciences ,Functional diversity ,0302 clinical medicine ,CRISPR-Associated Protein 9 ,Animals ,Humans ,CRISPR ,Rna targeting ,030304 developmental biology ,Gene Editing ,0303 health sciences ,RNA ,Genetic Therapy ,Cell Biology ,Cell biology ,Metagenomics ,030220 oncology & carcinogenesis ,Metagenome ,CRISPR-Cas Systems ,Genetic Engineering ,RNA, Guide, Kinetoplastida - Abstract
Deployment of RNA-guided DNA endonuclease CRISPR-Cas technology has led to radical advances in biology. As the functional diversity of CRISPR-Cas and parallel systems is further explored, RNA manipulation is emerging as a powerful mode of CRISPR-based engineering. In this Perspective, we chart progress in the RNA-targeting CRISPR-Cas (RCas) field and illustrate how continuing evolution in scientific discovery translates into applications for RNA biology and insights into mysteries, obstacles, and alternative technologies that lie ahead.
- Published
- 2020
35. In vitro Evaluation of Bis-3-chloropiperidines as RNA Modulators Targeting TAR and TAR-protein Interaction
- Author
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Alice Sosic, Giulia Olivato, Caterina Carraro, Richard Göttlich, Dan Fabris, and Barbara Gatto
- Subjects
QH301-705.5 ,medicinal_chemistry ,Organic Chemistry ,RNA targeting ,RNA-based interactions ,bis-3-chloropiperidines ,General Medicine ,Nucleocapsid Proteins ,Article ,Catalysis ,Computer Science Applications ,Inorganic Chemistry ,Chemistry ,Piperidines ,HIV-1 ,Nucleic Acid Conformation ,RNA, Viral ,Biology (General) ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,Bis-3-chloropiperidines ,Spectroscopy - Abstract
After a long limbo, RNA has gained its credibility as a druggable target, fully earning its de-served role in the next-generation area of pharmaceutical R&D. We have recently probed the Trans-Activation Response element (TAR), a RNA stem–bulge–loop domain of the HIV-1 genome with bis-3-chloropiperidines (B-CePs), and revealed the compounds unique behavior in stabiliz-ing TAR structure, thus impairing in vitro the chaperone activity of the HIV-1 nucleocapsid (NC) protein. Seeking to elucidate the determinants of B-CePs inhibition, we have further characterized here their effects on the target TAR and its NC recognition, while developing quantitative analyti-cal approaches for the study of multicomponent RNA-based interactions.
- Published
- 2021
36. New Type III CRISPR variant and programmable RNA targeting tool: Oh, thank heaven for Cas7-11
- Author
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Michael P. Terns and Ryan J. Catchpole
- Subjects
Trans-activating crRNA ,Gene knockdown ,CRISPR ,RNA ,Cell Biology ,Computational biology ,RNA Cleavage ,Biology ,Rna targeting ,Molecular Biology ,Article - Abstract
Ozcan et al. (2021) and van Beljouw et al. (2021) characterize a novel Type III-E CRISPR-Cas subtype, composed of a single polypeptide with crRNA processing and sequence-specific RNA cleavage activities, that provides a new RNA knockdown tool for mammalian cells with fewer off-target effects than current technologies.
- Published
- 2021
37. RNA Targeting in Inherited Neuromuscular Disorders: Novel Therapeutic Strategies to Counteract Mis-Splicing
- Author
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Gabriele Ferrante, Maria Paola Paronetto, Veronica Verdile, and Gloria Guizzo
- Subjects
Neuromuscular disease ,QH301-705.5 ,Neuromuscular Junction ,Review ,Bioinformatics ,Models, Biological ,alternative splicing ,Atrophy ,Humans ,Medicine ,Biology (General) ,Muscle, Skeletal ,Rna targeting ,Oligonucleotide ,business.industry ,Alternative splicing ,RNA-based therapies ,RNA ,Neuromuscular Diseases ,General Medicine ,neuromuscular disease ,medicine.disease ,MRNA metabolism ,RNA splicing ,business - Abstract
Neuromuscular disorders represent multifaceted abnormal conditions, with little or no cure, leading to patient deaths from complete muscle wasting and atrophy. Despite strong efforts in the past decades, development of effective treatments is still urgently needed. Advent of next-generation sequencing technologies has allowed identification of novel genes and mutations associated with neuromuscular pathologies, highlighting splicing defects as essential players. Deciphering the significance and relative contributions of defective RNA metabolism will be instrumental to address and counteract these malignancies. We review here recent progress on the role played by alternative splicing in ensuring functional neuromuscular junctions (NMJs), and its involvement in the pathogenesis of NMJ-related neuromuscular disorders, with particular emphasis on congenital myasthenic syndromes and muscular dystrophies. We will also discuss novel strategies based on oligonucleotides designed to bind their cognate sequences in the RNA or targeting intermediary of mRNA metabolism. These efforts resulted in several chemical classes of RNA molecules that have recently proven to be clinically effective, more potent and better tolerated than previous strategies.
- Published
- 2021
38. Photodynamic Therapy with Tumor Cell Discrimination through RNA-Targeting Ability of Photosensitizer
- Author
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Xiaoyi Jin, Yuan Xu, Xiuqin Ma, Yang Tan, Miao Li, and Ye Tian
- Subjects
cyanine dyes ,cancer cell discrimination ,photosensitizer ,medicine.medical_treatment ,Pharmaceutical Science ,Organic chemistry ,Photodynamic therapy ,Tumor cells ,Enhanced permeability and retention effect ,Article ,Analytical Chemistry ,QD241-441 ,RNA targeting ,Cell Line, Tumor ,Neoplasms ,Drug Discovery ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Photosensitizer ,Molecular Targeted Therapy ,Physical and Theoretical Chemistry ,Low-Level Light Therapy ,Rna targeting ,Mice, Inbred BALB C ,Photosensitizing Agents ,Chemistry ,Cancer ,RNA ,medicine.disease ,Xenograft Model Antitumor Assays ,Photochemotherapy ,Chemistry (miscellaneous) ,Cancer cell ,COS Cells ,Injections, Intravenous ,Cancer research ,MCF-7 Cells ,Molecular Medicine ,Female - Abstract
Photodynamic therapy (PDT) represents an effective treatment to cure cancer. The targeting ability of the photosensitizer is of utmost importance. Photosensitizers that discriminate cancer cells can avoid the killing of normal cells and improve PDT efficacy. However, the design and synthesis of photosensitizers conjugated with a recognition unit of cancer cell markers is complex and may not effectively target cancer. Considering that the total RNA content in cancer cells is commonly higher than in normal cells, this study has developed the photosensitizer QICY with RNA-targeting abilities for the discrimination of cancer cells. QICY was specifically located in cancer cells rather than normal cells due to their stronger electrostatic interactions with RNA, thereby further improving the PDT effects on the cancer cells. After intravenous injection into mice bearing a xenograft tumor, QICY accumulated into the tumor location through the enhanced permeability and retention effect, automatically targeted cancer cells under the control of RNA, and inhibited tumor growth under 630 nm laser irradiation without obvious side effects. This intelligent photosensitizer with RNA-targeting ability not only simplifies the design and synthesis of cancer-cell-targeting photosensitizers but also paves the way for the further development of highly efficient PDTs.
- Published
- 2021
39. CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.
- Author
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Song, Jinghui, Dong, Liting, Sun, Hanxiao, Luo, Nan, Huang, Qiang, Li, Kai, Shen, Xiaowen, Jiang, Zhe, Lv, Zhicong, Peng, Luxin, Zhang, Meifang, Wang, Kun, Liu, Ke, Hong, Jiaxu, and Yi, Chengqi
- Subjects
- *
STOP codons , *RNA , *NONSENSE mutation , *PSEUDOURIDINE , *URIDINE - Abstract
Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics. [Display omitted] • RESTART relies on guide snoRNA to revert PTC-induced translation termination • The minor DKC1-isoform3 greatly increases the RNA-editing efficiency of RESTART • RESTART enables efficient pseudouridylation in cell lines and primary cells • RESTART mediates functional PTC readthrough in disease-relevant contexts Song et al. developed a programmable RNA base editor, termed RESTART, relying on guide snoRNA to mediate site-specific pseudouridylation in mammalian cells. RESTART was applied to nonsense mutations in multiple disease contexts to achieve functional PTC readthrough. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
40. Targeting RNA with small molecules: lessons learned from Xist RNA.
- Author
-
Nickbarg EB, Spencer KB, Mortison JD, and Lee JT
- Subjects
- Humans, X Chromosome Inactivation, RNA, Untranslated genetics, Proteins genetics, RNA, Long Noncoding metabolism
- Abstract
Although more than 98% of the human genome is noncoding, nearly all drugs on the market target one of about 700 disease-related proteins. However, an increasing number of diseases are now being attributed to noncoding RNA and the ability to target them would vastly expand the chemical space for drug development. We recently devised a screening strategy based upon affinity-selection mass spectrometry and succeeded in identifying bioactive compounds for the noncoding RNA prototype, Xist. One such compound, termed X1, has drug-like properties and binds specifically to the RepA motif of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that X1 changes the conformation of RepA in solution, thereby explaining the displacement of cognate interacting protein factors (PRC2 and SPEN) and inhibition of X-chromosome inactivation. In this Perspective, we discuss lessons learned from these proof-of-concept experiments and suggest that RNA can be systematically targeted by drug-like compounds to disrupt RNA structure and function., (© 2023 Nickbarg et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2023
- Full Text
- View/download PDF
41. RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences.
- Author
-
Derksen, Merel, Mertens, Vicky, and Pruijn, Ger J. M.
- Subjects
- *
RNA analysis , *ESCHERICHIA coli , *GENE expression - Abstract
The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
42. Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1
- Author
-
Ondono, Raul, Lirio, Ángel, Elvira, Carlos, Álvarez Marimon, Elena, Provenzano, Claudia, Cardinali, Beatrice, Pérez-Alonso, Manuel, Peralvarez-Marin, Alex, Borrell, José I., Falcone, Germana, Estrada-Tejedor, Roger, Ondono, Raul, Lirio, Ángel, Elvira, Carlos, Álvarez Marimon, Elena, Provenzano, Claudia, Cardinali, Beatrice, Pérez-Alonso, Manuel, Peralvarez-Marin, Alex, Borrell, José I., Falcone, Germana, and Estrada-Tejedor, Roger
- Abstract
Altres ajuts: Obra Social "La Caixa", Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3' untranslated region (3'UTR). The intrinsic complexity and lack of crystallographic data makes noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequester many essential alternative splicing factors such as Muscleblind-like 1 (MBNL1). Most DM1 phenotypic features stem from the reduced availability of free MBNL1 and therefore many therapeutic efforts are focused on recovering its normal activity. For that purpose, herein we present pyrido[2,3- d ]pyrimidin-7-(8 H)-ones, a privileged scaffold showing remarkable biological activity against many targets involved in human disorders including cancer and viral diseases. Their combination with a flexible linker meets the requirements to stabilise DM1 toxic transcripts, and therefore, enabling the release of MBNL1. Therefore, a set of novel pyrido[2,3- d ]pyrimidin-7-(8 H)-ones derivatives (1a - e) were obtained using click chemistry. 1a exerted over 20% MBNL1 recovery on DM1 toxic RNA activity in primary cell biology studies using patient-derived myoblasts. 1a promising anti DM1 activity may lead to subsequent generations of ligands, highlighting a new affordable treatment against DM1.
- Published
- 2021
43. Controlling Site-Directed RNA Editing by Chemically Induced Dimerization
- Author
-
Ruth Lappalainen, Anna S. Stroppel, and Thorsten Stafforst
- Subjects
Adenosine Deaminase ,Endogeny ,Computational biology ,medicine.disease_cause ,01 natural sciences ,Catalysis ,03 medical and health sciences ,Basic research ,RNA targeting ,medicine ,Rna targeting ,Glyceraldehyde 3-phosphate dehydrogenase ,030304 developmental biology ,0303 health sciences ,Mutation ,biology ,010405 organic chemistry ,Chemistry ,Communication ,Organic Chemistry ,chemically induced dimerization ,site-directed RNA editing ,RNA-Binding Proteins ,General Chemistry ,ADAR ,Communications ,0104 chemical sciences ,RNA editing ,biology.protein ,Chemically induced dimerization ,RNA ,RNA Editing ,Dimerization ,gibberellic acid - Abstract
Various RNA‐targeting approaches have been engineered to modify specific sites on endogenous transcripts, breaking new ground for a variety of basic research tools and promising clinical applications in the future. Here, we combine site‐directed adenosine‐to‐inosine RNA editing with chemically induced dimerization. Specifically, we achieve tight and dose‐dependent control of the editing reaction with gibberellic acid, and obtain editing yields up to 20 % and 44 % in the endogenous STAT1 and GAPDH transcript in cell culture. Furthermore, the disease‐relevant MECP2 R106Q mutation was repaired with editing yields up to 42 %. The introduced principle will enable new applications where temporal or spatiotemporal control of an RNA‐targeting mechanism is desired., RNA editing: Site‐directed adenosine‐to‐inosine RNA editing was engineered to be under control of the plant hormone gibberellic acid, applying the mechanism of chemically induced dimerization. Tight control and editing yields up to 44 % where achieved on endogenous targets in human cell culture.
- Published
- 2021
44. Improving CRISPR‐Cas‐mediated RNA targeting and gene editing using SPLCV replicon‐based expression vectors in Nicotiana benthamiana
- Author
-
Houjun Sun, Xiao Wang, Zongyun Li, Qiang Liang, Chengling Zhang, Yicheng Yu, Qiang Li, Qinghe Cao, Yiping Xie, Jian Sun, Xiaofeng Bian, Daifu Ma, and Weichi Wang
- Subjects
Expression vector ,LbCas12a ,Nicotiana benthamiana ,SpCas9 ,Plant Science ,Computational biology ,Biology ,Brief Communication ,biology.organism_classification ,RNA targeting and gene editing efficiency ,deconstructed SPLCV strategy ,Genome editing ,CRISPR ,LwaCas13a ,Replicon ,Brief Communications ,Rna targeting ,Agronomy and Crop Science ,Biotechnology - Published
- 2020
45. miRNA inhibition by proximity-enabled Dicer inactivation
- Author
-
Hao Yan and Fu-Sen Liang
- Subjects
Ribonuclease III ,chemistry.chemical_classification ,biology ,food and beverages ,Small molecule ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Small Molecule Libraries ,MicroRNAs ,Enzyme ,Gene Expression Regulation ,chemistry ,Mirna expression ,microRNA ,biology.protein ,Humans ,RNA ,Enzyme Inhibitors ,MiRNA biogenesis ,Rna targeting ,Molecular Biology ,Dicer - Abstract
microRNAs (miRNAs) are considered as master regulators of biological processes. Dysregulation of miRNA expression has been implicated in many human diseases. Driven by the key biological roles and the therapeutic potential, developing methods for miRNA regulation has become an intense research area. Due to favorable pharmacological properties, small molecule-based miRNA inhibition emerges as a promising strategy and significant progresses have been made. However, it remains challenging to regulate miRNA using small molecules because of the inherent difficulty in RNA targeting and inhibition. Herein we outline the workflow of generating bifunctional small molecule inhibitors blocking miRNA biogenesis through proximity-enabled inactivation of Dicer, an enzyme required for the processing of precursor miRNA (pre-miRNA) into mature miRNA. By conjugating a weak Dicer inhibitor with a pre- miRNA binder, the inhibitor can be delivered to the Dicer processing site associated with the targeted pre-miRNA, and as a result inhibiting Dicer-mediated pre-miRNA processing. This protocol can be applicable in producing bifunctional inhibitors for different miRNAs.
- Published
- 2019
46. In Silico Targeting of the Long Noncoding RNA MALAT1
- Author
-
May Khanna, Vijay Gokhale, Victor G. Miranda, Niloufar Mollasalehi, and Liberty François-Moutal
- Subjects
MALAT1 ,010405 organic chemistry ,In silico ,Organic Chemistry ,RNA ,Negative control ,Computational biology ,Biology ,01 natural sciences ,Biochemistry ,Long non-coding RNA ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Drug Discovery ,Transfer RNA ,Gene expression ,Rna targeting - Abstract
[Image: see text] RNA targeting has gained traction over the past decade. It has become clear that dysregulation of RNA can be linked to many diseases, leading to a need for new scaffolds recognizing RNA specifically. Long noncoding RNAs are emerging as key controllers of gene expression and potential therapeutic targets. However, traditional targeting methods have overwhelmingly been focused on proteins. In this study, we used a protein computational tool and found several possible targetable pockets in a structurally characterized long noncoding RNA, MALAT1. Screening against those identified pockets revealed several hit compounds. We tested the binding of those compounds to MALAT1 RNA and tRNA as a negative control, using SPR. While several compounds were nonspecific binders, others were able to recognize MALAT1 specifically. One of them, MTC07, has an apparent affinity of 400.2 ± 14.4 μM. Although it has weak affinity, MTC07 is the first compound targeting MALAT1 originating from in silico docking.
- Published
- 2021
47. Supramolecular cylinders target bulge structures in the 5' UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication
- Author
-
Melidis, Lazaros, Davies, Scott, Coltman, Nicholas, Winczura, Kinga, Hodges, Nikolas J., Zania Stamataki, Grzechnik, Pawel, Hannon, Michael J., and Lazaros Melidis
- Subjects
RNA targeting ,viruses ,coordination chemistry ,sars-cov2 ,Molecular Dynamics ,UTR - Abstract
Supplementary and raw data on computational aspects of paper Titled;Supramolecular cylinders target bulge structures in the 5’ UTR of the RNA genome of SARS-CoV-2 and inhibit viral replication.
- Published
- 2021
- Full Text
- View/download PDF
48. Evaluation of a Trio Toscana Virus Real-Time RT-PCR Assay Targeting Three Genomic Regions within Nucleoprotein Gene
- Author
-
Xavier de Lamballerie, Sara Sanbonmatsu-Gámez, Irene Pedrosa-Corral, Rémi N. Charrel, Alessandra Falchi, Mercedes Pérez-Ruiz, Laura Pezzi, Laurence Thirion, [Thirion,L, Pezzi,L, de Lamballerie,X, Charrel,RN] Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207-IHU Méditerranée Infection), Marseille, France. [Pezzi,L, Falchi,A] UR7310, Laboratoire de Virologie, Université de Corse-Inserm, Corte, France. [Pedrosa-Corral,I, Sanbonmatsu-Gamez,S, Perez-Ruiz,M] Servicio de Microbiología, Hospital Universitario Virgen de las Nieves, Instituto de Investigación Biosanitaria ibs.Granada, Granada, Spain., This study was partially funded by (i) the 'European Virus Archive Global' (EVA GLOBAL) project H2020-INFRAIA-2019 program, Project No 871029, and (ii) 'Emerging Viral Diseases—Expert Laboratory Network' (EVDLabNET).
- Subjects
Microbiology (medical) ,Phlebovirus ,Organisms::Viruses::RNA Viruses::Bunyaviridae::Phlebovirus::Sandfly fever Naples virus [Medical Subject Headings] ,Information Science::Information Science::Medical Informatics::Medical Informatics Applications::Information Storage and Retrieval::Data Mining [Medical Subject Headings] ,In silico ,lcsh:Medicine ,Phenuiviridae ,Diseases::Bacterial Infections and Mycoses::Central Nervous System Infections [Medical Subject Headings] ,Biology ,Mediterranean ,Arbovirus ,Article ,Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings] ,03 medical and health sciences ,Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::RNA [Medical Subject Headings] ,medicine ,diagnostics ,Immunology and Allergy ,Meningitis ,Rna targeting ,Diagnostics ,Molecular Biology ,Gene ,030304 developmental biology ,0303 health sciences ,General Immunology and Microbiology ,030306 microbiology ,Toscana virus ,Diagnóstico ,lcsh:R ,Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Nucleoproteins [Medical Subject Headings] ,meningitis ,Phlebotomus ,medicine.disease ,biology.organism_classification ,Virology ,3. Good health ,Nucleoprotein ,Infectious Diseases ,Real-time polymerase chain reaction ,arbovirus ,Sand fly ,Psychodidae ,sand fly - Abstract
Toscana virus (TOSV) can cause central nervous system infections in both residents of and travelers to Mediterranean countries. Data mining identified three real-time RT-qPCR assays for detecting TOSV RNA targeting non-overlapping regions in the nucleoprotein gene. Here, they were combined to create a multi-region assay named Trio TOSV RT-qPCR consisting of six primers and three probes. In this study, (i) we evaluated in silico the three RT-qPCR assays available in the literature for TOSV detection, (ii) we combined the three systems to create the Trio TOSV RT-qPCR, (iii) we assessed the specificity and sensitivity of the three monoplex assays versus the Trio TOSV RT-qPCR assay, and (iv) we compared the performance of the Trio TOSV RT-qPCR assay with one of the reference monoplex assays on clinical samples. In conclusion, the Trio TOSV RT-qPCR assay performs equally or better than the three monoplex assays, therefore, it provides a robust assay that can be used for both research and diagnostic purposes.
- Published
- 2021
49. Applications of Cas9 as an RNA-programmed RNA-binding protein.
- Author
-
Nelles, David A., Fang, Mark Y., Aigner, Stefan, and Yeo, Gene W.
- Subjects
- *
CARRIER proteins , *STREPTOCOCCUS pyogenes , *GENOME editing , *GENETIC regulation , *NUCLEOTIDE sequence , *GENE expression , *SYNTHETIC biology - Abstract
The Streptococcus pyogenes CRISPR-Cas system has gained widespread application as a genome editing and gene regulation tool as simultaneous cellular delivery of the Cas9 protein and guide RNAs enables recognition of specific DNA sequences. The recent discovery that Cas9 can also bind and cleave RNA in an RNA-programmable manner indicates the potential utility of this system as a universal nucleic acid-recognition technology. RNA-targeted Cas9 (RCas9) could allow identification and manipulation of RNA substrates in live cells, empowering the study of cellular gene expression, and could ultimately spawn patient- and disease-specific diagnostic and therapeutic tools. Here we describe the development of RCas9 and compare it to previous methods for RNA targeting, including engineered RNA-binding proteins and other types of CRISPR-Cas systems. We discuss potential uses ranging from live imaging of transcriptional dynamics to patient-specific therapies and applications in synthetic biology. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
50. CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos
- Author
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Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fondo Nacional de Desarrollo Científico, Tecnológico y de Información Tecnológica (Perú), Kushawah, Gopal, Abugattas-Nuñez del Prado, Joaquin A., Martínez-Morales, Juan Ramón, DeVore, Michelle L., Hassan, Huzaifa, Moreno-Sánchez, Ismael, Tomás-Gallardo, Laura, Díaz Moscoso, Alejandro, Monges, Dahiana E., Guelfo, Javier R., Theune, William C., Brannan, Emry O., Wang, Wei, Corbin, Timothy J., Moran, Andrea M., Sánchez Alvarado, Alejandro, Málaga-Trillo, Edward, Takacs, Carter M., Bazzini, Ariel A., Moreno-Mateos, Miguel A., Ministerio de Economía y Competitividad (España), Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fondo Nacional de Desarrollo Científico, Tecnológico y de Información Tecnológica (Perú), Kushawah, Gopal, Abugattas-Nuñez del Prado, Joaquin A., Martínez-Morales, Juan Ramón, DeVore, Michelle L., Hassan, Huzaifa, Moreno-Sánchez, Ismael, Tomás-Gallardo, Laura, Díaz Moscoso, Alejandro, Monges, Dahiana E., Guelfo, Javier R., Theune, William C., Brannan, Emry O., Wang, Wei, Corbin, Timothy J., Moran, Andrea M., Sánchez Alvarado, Alejandro, Málaga-Trillo, Edward, Takacs, Carter M., Bazzini, Ariel A., and Moreno-Mateos, Miguel A.
- Abstract
Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.
- Published
- 2020
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