scholarly journals RNA-Targeting CRISPR–Cas Systems and Their Applications

2020 ◽  
Vol 21 (3) ◽  
pp. 1122 ◽  
Author(s):  
Michal Burmistrz ◽  
Kamil Krakowski ◽  
Agata Krawczyk-Balska
Keyword(s):  
Molecular Biology ◽  
Current Knowledge ◽  
Type Ii ◽  
Rna Molecules ◽  
Dna Targeting ◽  
Modern Molecular Biology ◽  
Rna Targeting ◽  
New Research ◽  
Target Rna ◽  
Research Tools

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR–Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR–Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.

scholarly journals RNA-dependent RNA targeting by CRISPR-Cas9

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Steven C Strutt ◽  
Rachel M Torrez ◽  
Emine Kaya ◽  
Oscar A Negrete ◽  
Jennifer A Doudna
Keyword(s):  
Type Ii ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Ability To Act ◽  
Protospacer Adjacent Motif ◽  
Rna Targeting ◽  
Target Rna ◽  
Dsdna Binding ◽  
Rna Phage

Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.

scholarly journals A nucleus-like compartment shields bacteriophage DNA from CRISPR-Cas and restriction nucleases

2018 ◽  
Author(s):  
Senén D. Mendoza ◽  
Joel D. Berry ◽  
Eliza S. Nieweglowska ◽  
Lina M. Leon ◽  
David A. Agard ◽  
...  
Keyword(s):  
Type I ◽  
Type Ii ◽  
Immune Systems ◽  
Dna Targeting ◽  
Rna Targeting ◽  
Immune Pathways ◽  
Type V ◽  
Restriction Modification

All viruses require strategies to inhibit or evade the immunity pathways of cells they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid nucleic-acid targeting immune pathways such as CRISPR-Cas and restriction endonucleases to replicate efficiently1. Here, we show that a jumbo phage infecting Pseudomonas aeruginosa, phage ΦKZ, is resistant to many immune systems in vivo, including CRISPR-Cas3 (Type I-C), Cas9 (Type II-A), Cas12 (Cpf1, Type V-A), and Type I restriction-modification (R-M) systems. We propose that ΦKZ utilizes a nucleus-like shell to protect its DNA from attack. Supporting this, we demonstrate that Cas9 is able to cleave ΦKZ DNA in vitro, but not in vivo and that Cas9 is physically occluded from the shell assembled by the phage during infection. Moreover, we demonstrate that the Achilles heel for this phage is the mRNA, as translation occurs outside of the shell, rendering the phage sensitive to the RNA targeting CRISPR-Cas enzyme, Cas13a (C2c2, Type VI-A). Collectively, we propose that the nucleus-like shell assembled by jumbo phages enables potent, broad spectrum evasion of DNA-targeting nucleases.

Beyond DNA-targeting in Cancer Chemotherapy. Emerging Frontiers- A Review

2020 ◽  
Vol 20 ◽  
Author(s):  
S.N Mbugua ◽  
L.W Njenga ◽  
R.A Odhiambo ◽  
S.O Wandiga ◽  
M.O Onani
Keyword(s):  
Superparamagnetic Iron Oxide ◽  
Active Centre ◽  
Dna Targeting ◽  
Advantages And Disadvantages ◽  
Cancer Pathogenesis ◽  
Ongoing Work ◽  
Target Dna ◽  
Anti Cancer ◽  
New Research ◽  
Innovative Drugs

: Modern anticancer drugs target DNA specifically on rapid division of malignant cells. One downside of this approach is that they also target other rapidly dividing healthy cells such as those involved in hair growth leading to serious toxic side effects and hair loss. Therefore, it would be better to develop novel agents that address cellular signalling mechanisms unique to cancerous cells, and new research is now focussing on such approaches. Although the classical chemotherapy area involving DNA as the set target continues to produce important findings, nevertheless, a distinctly discernible emerging trend is the divergence from the cisplatin operation model that uses the metal as the primary active centre of the drug. Many successful anti-cancer drugs present are associated with elevated toxicity levels. Cancers also develop immunity against most therapies and the area of cancer research can therefore be seen as an area with a high unaddressed need. Hence, ongoing work into cancer pathogenesis is important to create accurate preclinical tests which can contribute to the development of innovative drugs to manage and treat cancer. Some of the emergent frontiers utilizing different approaches include nanoparticles delivery, use of quantum dots, metal complexes, tumour ablation, magnetic hypothermia and hyperthermia by use of Superparamagnetic Iron oxide Nanostructures, pathomics and radiomics, laser surgery and exosomes. This review summarizes these new approaches in good detail giving critical views with necessary comparisons. It also delves into what they carry for the future including their advantages and disadvantages.

scholarly journals Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Science ◽  
2021 ◽  
pp. eabe7106
Author(s):  
Chunlei Jiao ◽  
Sahil Sharma ◽  
Gaurav Dugar ◽  
Natalia L. Peeck ◽  
Thorsten Bischler ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Genetic Material ◽  
Simultaneous Detection ◽  
Type Ii ◽  
Rna Detection ◽  
Single Base ◽  
Dna Targeting ◽  
Single Base Resolution ◽  
Rna Complexes

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In Type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of “noncanonical” crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA-of-interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished SARS-CoV-2 and its D614G variant with single-base resolution in patient samples.

scholarly journals Molecular biology approaches in bioadhesion research

2014 ◽  
Vol 5 ◽  
pp. 983-993 ◽  
Author(s):  
Marcelo Rodrigues ◽  
Birgit Lengerer ◽  
Thomas Ostermann ◽  
Peter Ladurner
Keyword(s):  
Molecular Biology ◽  
Biological Function ◽  
Molecular Approach ◽  
Research Groups ◽  
Blast Search ◽  
Search Facility ◽  
New Research ◽  
And Function ◽  
Functional Analyses

The use of molecular biology tools in the field of bioadhesion is still in its infancy. For new research groups who are considering taking a molecular approach, the techniques presented here are essential to unravelling the sequence of a gene, its expression and its biological function. Here we provide an outline for addressing adhesion-related genes in diverse organisms. We show how to gradually narrow down the number of candidate transcripts that are involved in adhesion by (1) generating a transcriptome and a differentially expressed cDNA list enriched for adhesion-related transcripts, (2) setting up a BLAST search facility, (3) perform an in situ hybridization screen, and (4) functional analyses of selected genes by using RNA interference knock-down. Furthermore, latest developments in genome-editing are presented as new tools to study gene function. By using this iterative multi-technologies approach, the identification, isolation, expression and function of adhesion-related genes can be studied in most organisms. These tools will improve our understanding of the diversity of molecules used for adhesion in different organisms and these findings will help to develop innovative bio-inspired adhesives.

Analysis of Aluminium Beam-to-Column Joints by the Component Method: Existing Studies and Research Needs

2016 ◽  
Vol 710 ◽  
pp. 409-414 ◽  
Author(s):  
Gianfranco De Matteis ◽  
Giuseppe Brando
Keyword(s):  
Current Knowledge ◽  
Component Method ◽  
The Past ◽  
Research Fields ◽  
Numerical Studies ◽  
Current State ◽  
Moment Resisting ◽  
New Research ◽  
Steel Joints

This paper aims at providing an overview on the current state of the art and on possible future developments concerning the component method implementation for the classification of beam-to-column joints belonging to aluminum moment resisting frames.After a brief discussion on the component method theoretical bases, developed in the past to give a feasible calculation procedure for steel joints, recent experimental and numerical studies, carried out for investigating some aluminum components, are presented and discussed. In particular strengths and weaknesses of the current knowledge are put into evidence, also in light of the peculiarities that make aluminum alloys different from steel. The launch of new research fields, aimed at pursuing an update of the current codes dealing with aluminum structures, is therefore proposed.

scholarly journals Systematizing Genome Privacy Research: A Privacy-Enhancing Technologies Perspective

2019 ◽  
Vol 2019 (1) ◽  
pp. 87-107 ◽  
Author(s):  
Alexandros Mittos ◽  
Bradley Malin ◽  
Emiliano De Cristofaro
Keyword(s):  
Data Privacy ◽  
Online Survey ◽  
Current Knowledge ◽  
Genomic Data ◽  
Well Being ◽  
Security And Privacy ◽  
Genome Data ◽  
Privacy Enhancing Technologies ◽  
Cost Efficient ◽  
New Research

Abstract Rapid advances in human genomics are enabling researchers to gain a better understanding of the role of the genome in our health and well-being, stimulating hope for more effective and cost efficient healthcare. However, this also prompts a number of security and privacy concerns stemming from the distinctive characteristics of genomic data. To address them, a new research community has emerged and produced a large number of publications and initiatives. In this paper, we rely on a structured methodology to contextualize and provide a critical analysis of the current knowledge on privacy-enhancing technologies used for testing, storing, and sharing genomic data, using a representative sample of the work published in the past decade. We identify and discuss limitations, technical challenges, and issues faced by the community, focusing in particular on those that are inherently tied to the nature of the problem and are harder for the community alone to address. Finally, we report on the importance and difficulty of the identified challenges based on an online survey of genome data privacy experts.

scholarly journals Historical and legal discourse in the context of classical and postclassical legal understanding

2021 ◽  
pp. 106-111
Author(s):  
Tetiana Bondaruk
Keyword(s):  
Legal System ◽  
Legal Discourse ◽  
Radical Change ◽  
Legal Environment ◽  
Stage Of Development ◽  
Starting Point ◽  
Legal Space ◽  
New Research ◽  
Legal Understanding ◽  
Research Tools

Іntroduction. Historical and legal science, as well as the science of law in general, is acutely faced with challenges related to the new stage of development of humanities knowledge and the corresponding change of research paradigm that occurs during the struggle between classical and nonclassical (postclassical) types of legal understanding Тhe aim of the article. Тhese processes need to be understood and "adapted" in particular in the historical and legal discourse. In particular, it is proposed to analyze the phenomenon of deformation of the phenomenon of law, and the resulting differentiation of the subject, in particular in historical and legal research, and the coherence of research tools offered within the classical and nonclassical types of legal understanding Results. Modern methodological research is a natural reaction of the domestic legal process to the dominance of the monistic materialist approach to the study of legal phenomena, which actualizes anthropological and axiological approaches. Both anthropologization and axiologization of law cause the deformalization of the phenomenon of law, creating a conceptual In the light of the above, it seems important to consider in relation to the relationship such concepts as legal reality (historical and legal reality), legal life, legal system as central, and legal space, legal field, legal environment as peripheral. At the same time, attention is drawn to the normative nature of the legal system, the ontological nature of legal reality, the inconsistency of legal life as a starting point in the choice of methodological tools. Introduction to the historical and legal discourse of «ontological metaphors»: legal communication, legal event, legal life, legal space, legal field, legal environment, etc., will activate the intersubjective model of knowledge of law as a sociocultural phenomenon, draw attention to the dynamics of law, using an arsenal of non-classical methodology. Conclusions. An overview of some trends that lead to a change in the object and subject of jurisprudence shows a radical change in its methodology, which should form research tools to answer new research questions. This process is part of the process of modern «cultivation» of integrated thinking as opposed to or in addition to analytical and systemic, which is characterized by consideration of reality in mechanistic categories, and, being irreversible, requires appropriate historical and legal reflections

scholarly journals Cooperation between CRISPR-Cas types enables adaptation in an RNA-targeting system

2020 ◽  
Author(s):  
Ville Hoikkala ◽  
Janne Ravantti ◽  
César Díez-Villaseñor ◽  
Marja Tiirola ◽  
Rachel A. Conrad ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Flavobacterium Columnare ◽  
Immune Systems ◽  
Dna Targeting ◽  
B Locus ◽  
Rna Targeting ◽  
In Trans ◽  
Apparent Variation ◽  
Dsdna Phage ◽  
Native Host

AbstractCRISPR-Cas immune systems adapt to new threats by acquiring spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition, despite apparent variation in spacer content between strains. It has been suggested that such loci may use acquisition machinery from co-occurring CRISPR-Cas systems. Here, using a lytic dsDNA phage, we observe spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient subtype VI-B locus and a complete subtype II-C locus. We characterize acquisition events in both loci and show that the RNA-targeting VI-B locus acquires spacers in trans using acquisition machinery from the DNA-targeting II-C locus. Our observations reinforce the concept of modularity in CRISPR-Cas systems and raise further questions regarding plasticity of adaptation modules.

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