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 Packaging of Genomic RNA in Positive-Sense Single-Stranded RNA Viruses: A Complex Story

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 253 ◽  
Author(s):  
Mauricio Comas-Garcia
Keyword(s):  
Rna Viruses ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Dna And Rna ◽  
Relative Contribution ◽  
Double Stranded Dna ◽  
Positive Sense ◽  
Infectious Cycle

The packaging of genomic RNA in positive-sense single-stranded RNA viruses is a key part of the viral infectious cycle, yet this step is not fully understood. Unlike double-stranded DNA and RNA viruses, this process is coupled with nucleocapsid assembly. The specificity of RNA packaging depends on multiple factors: (i) one or more packaging signals, (ii) RNA replication, (iii) translation, (iv) viral factories, and (v) the physical properties of the RNA. The relative contribution of each of these factors to packaging specificity is different for every virus. In vitro and in vivo data show that there are different packaging mechanisms that control selective packaging of the genomic RNA during nucleocapsid assembly. The goals of this article are to explain some of the key experiments that support the contribution of these factors to packaging selectivity and to draw a general scenario that could help us move towards a better understanding of this step of the viral infectious cycle.

scholarly journals Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases

2017 ◽  
Author(s):  
Ole Niewoehner ◽  
Carmela Garcia-Doval ◽  
Jakob T. Rostøl ◽  
Christian Berk ◽  
Frank Schwede ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Rna Binding ◽  
Second Messengers ◽  
Allosteric Activation ◽  
Conceptual Similarity ◽  
Type Iii ◽  
Rna Transcripts ◽  
Rna Targeting ◽  
Target Rna

ABSTRACTIn many prokaryotes, type III CRISPR–Cas systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex that possesses dual RNase and DNase activities. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the type III interference complex, the Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to a hitherto unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

scholarly journals PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jochem N. A. Vink ◽  
Jan H. L. Baijens ◽  
Stan J. J. Brouns
Keyword(s):  
Large Scale ◽  
Open Reading Frames ◽  
Type I ◽  
Metagenomic Sequence ◽  
Template Strand ◽  
Dna Targeting ◽  
Dna And Rna ◽  
Protospacer Adjacent Motif ◽  
Wide Range ◽  
Rna Targeting

Abstract Background The adaptive CRISPR-Cas immune system stores sequences from past invaders as spacers in CRISPR arrays and thereby provides direct evidence that links invaders to hosts. Mapping CRISPR spacers has revealed many aspects of CRISPR-Cas biology, including target requirements such as the protospacer adjacent motif (PAM). However, studies have so far been limited by a low number of mapped spacers in the database. Results By using vast metagenomic sequence databases, we map approximately one-third of more than 200,000 unique CRISPR spacers from a variety of microbes and derive a catalog of more than two hundred unique PAM sequences associated with specific CRISPR-Cas subtypes. These PAMs are further used to correctly assign the orientation of CRISPR arrays, revealing conserved patterns between the last nucleotides of the CRISPR repeat and PAM. We could also deduce CRISPR-Cas subtype-specific preferences for targeting either template or coding strand of open reading frames. While some DNA-targeting systems (type I-E and type II systems) prefer the template strand and avoid mRNA, other DNA- and RNA-targeting systems (types I-A and I-B and type III systems) prefer the coding strand and mRNA. In addition, we find large-scale evidence that both CRISPR-Cas adaptation machinery and CRISPR arrays are shared between different CRISPR-Cas systems. This could lead to simultaneous DNA and RNA targeting of invaders, which may be effective at combating mobile genetic invaders. Conclusions This study has broad implications for our understanding of how CRISPR-Cas systems work in a wide range of organisms for which only the genome sequence is known.

scholarly journals Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Ville Hoikkala ◽  
Janne Ravantti ◽  
César Díez-Villaseñor ◽  
Marja Tiirola ◽  
Rachel A. Conrad ◽  
...  
Keyword(s):  
Bacterial Genome ◽  
Flavobacterium Columnare ◽  
Content Type ◽  
Immune Systems ◽  
Dna Cleaving ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Rna Targeting ◽  
Microbial Strains ◽  
Target Rna

ABSTRACT CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules. IMPORTANCE CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader’s genome. These fragments, called spacers, are integrated into a memory bank on the bacterial genome called the CRISPR array. The spacers allow for the recognition of the same invader upon subsequent infection. Most CRISPR-Cas systems target DNA, but recently, systems that exclusively target RNA have been discovered. RNA-targeting CRISPR-Cas systems often lack genes necessary for spacer acquisition, and it is thus unknown how new spacers are acquired and if they can be acquired from DNA phages. Here, we show that an RNA-targeting system “borrows” acquisition machinery from another CRISPR-Cas locus in the genome. Most new spacers in this locus are unable to target phage mRNA and are therefore likely redundant. Our results reveal collaboration between distinct CRISPR-Cas types and raise further questions on how other CRISPR-Cas loci may cooperate.

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 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.

scholarly journals Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA

Open Biology ◽  
2021 ◽  
Vol 11 (10) ◽  
Author(s):  
Megumi Endo ◽  
Jung In Kim ◽  
Narumi Aoki Shioi ◽  
Shigenori Iwai ◽  
Isao Kuraoka
Keyword(s):  
Arabidopsis Thaliana ◽  
Rna Editing ◽  
Homo Sapiens ◽  
Double Stranded Rna ◽  
Dna And Rna ◽  
Endonuclease V ◽  
Phosphodiester Bonds ◽  
Double Stranded Dna ◽  
Low Reaction Temperature

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli , whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana , the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3′ to inosine in single-strand RNA, at a low reaction temperature of 20–25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo .

In vivo glucose metabolism in obese and type II diabetic subjects with or without hypertension

Diabetes ◽  
1993 ◽  
Vol 42 (5) ◽  
pp. 764-772 ◽  
Author(s):  
E. Bonora ◽  
R. C. Bonadonna ◽  
S. Del Prato ◽  
G. Gulli ◽  
A. Solini ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Type Ii

scholarly journals Phenotypic Analysis of Mice Lacking the Tmprss2-Encoded Protease

2006 ◽  
Vol 26 (3) ◽  
pp. 965-975 ◽  
Author(s):  
Tom S. Kim ◽  
Cynthia Heinlein ◽  
Robert C. Hackman ◽  
Peter S. Nelson
Keyword(s):  
Serine Protease ◽  
Serine Proteases ◽  
Biological Activities ◽  
Type Ii ◽  
Phenotypic Analysis ◽  
Normal Prostate ◽  
Prostate Hyperplasia ◽  
Transmembrane Serine Protease

ABSTRACT Tmprss2 encodes an androgen-regulated type II transmembrane serine protease (TTSP) expressed highly in normal prostate epithelium and has been implicated in prostate carcinogenesis. Although in vitro studies suggest protease-activated receptor 2 may be a substrate for TMPRSS2, the in vivo biological activities of TMPRSS2 remain unknown. We generated Tmprss2 −/− mice by disrupting the serine protease domain through homologous recombination. Compared to wild-type littermates, Tmprss2 −/− mice developed normally, survived to adulthood with no differences in protein levels of prostatic secretions, and exhibited no discernible abnormalities in organ histology or function. Loss of TMPRSS2 serine protease activity did not influence fertility, reduce survival, result in prostate hyperplasia or carcinoma, or alter prostatic luminal epithelial cell regrowth following castration and androgen replacement. Lack of an observable phenotype in Tmprss2 −/− mice was not due to transcriptional compensation by closely related Tmprss2 homologs. We conclude that the lack of a discernible phenotype in Tmprss2 −/− mice suggests functional redundancy involving one or more of the type II transmembrane serine protease family members or other serine proteases. Alternatively, TMPRSS2 may contribute a specialized but nonvital function that is apparent only in the context of stress, disease, or other systemic perturbation.

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