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.

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 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 Single-Molecule Observation of Viral DNA Targeting by CRISPR/Cas Immune Systems

2013 ◽  
Vol 104 (2) ◽  
pp. 198a
Author(s):  
Samuel H. Sternberg ◽  
Sy Redding ◽  
Prashant Bhat ◽  
Martin Jinek ◽  
Blake Wiedenheft ◽  
...  
Keyword(s):  
Single Molecule ◽  
Viral Dna ◽  
Immune Systems ◽  
Dna Targeting

scholarly journals A target expression threshold dictates invader defense and autoimmunity by CRISPR-Cas13

2021 ◽  
Author(s):  
Elena Vialetto ◽  
Yanying Yu ◽  
Scott P. Collins ◽  
Katharina G. Wandera ◽  
Lars Barquist ◽  
...  
Keyword(s):  
Immune Activation ◽  
Rna Degradation ◽  
Lytic Bacteriophage ◽  
Immune Systems ◽  
Expression Levels ◽  
Additional Requirement ◽  
Genome Wide ◽  
A Genome ◽  
Rna Targeting ◽  
Target Transcript

Immune systems must recognize and clear foreign invaders without eliciting autoimmunity. CRISPR-Cas immune systems in prokaryotes manage this task by following two criteria: extensive guide:target complementarity and a defined target-flanking motif. Here we report an additional requirement for RNA-targeting CRISPR-Cas13 systems: expression of the target transcript exceeding a threshold. This finding is based on targeting endogenous non-essential transcripts, which rarely elicited dormancy through collateral RNA degradation. Instead, eliciting dormancy required over-expressing targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as the principal determinant of cytotoxic autoimmunity and revealed that the threshold shifts with the guide:target pair. This expression threshold ensured defense against a lytic bacteriophage yet allowed tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as a third criterion for immune activation by RNA-targeting CRISPR-Cas systems, buffering against autoimmunity and distinguishing pathogenic and benign invaders.

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 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.

Magnetic peptide nucleic acids for DNA targeting

2009 ◽  
pp. 6017 ◽  
Author(s):  
Giuseppe Prencipe ◽  
Stefano Maiorana ◽  
Paolo Verderio ◽  
Miriam Colombo ◽  
Paola Fermo ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Peptide Nucleic Acids ◽  
Dna Targeting

scholarly journals A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Nhan Huynh ◽  
Noah Depner ◽  
Raegan Larson ◽  
Kirst King-Jones
Keyword(s):  
Gene Expression ◽  
Nucleic Acids ◽  
High Efficiency ◽  
Ex Vivo ◽  
Transcriptional Level ◽  
Rna Targeting ◽  
Versatile Tool ◽  
Mitochondrial Transcripts ◽  
Overall Performance

AbstractAdvances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.

scholarly journals Spacer acquisition by Type III CRISPR–Cas system during bacteriophage infection of Thermus thermophilus

2020 ◽  
Vol 48 (17) ◽  
pp. 9787-9803
Author(s):  
Daria Artamonova ◽  
Karyna Karneyeva ◽  
Sofia Medvedeva ◽  
Evgeny Klimuk ◽  
Matvey Kolesnik ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Essential Gene ◽  
Thermus Thermophilus ◽  
Type Iii ◽  
Dna Targeting ◽  
Integral Number ◽  
Bacteriophage Infection ◽  
Rna Targeting ◽  
Infected Cells ◽  
Foreign Dna

Abstract Type III CRISPR–Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.

RNA-targeting low-molecular-weight fluorophores for nucleoli staining: synthesis, in silico modelling and cellular imaging

2021 ◽  
Author(s):  
Atanas Kurutos ◽  
Jasmina Nikodinovic Runic ◽  
Aleksandar M Veselinović ◽  
Jovana B. Veselinović ◽  
Fadhil Suliman Kamounah ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Molecular Docking ◽  
Nucleic Acids ◽  
In Silico ◽  
Photophysical Properties ◽  
Low Molecular Weight ◽  
Carbocyanine Dyes ◽  
Imaging Application ◽  
Rna Targeting ◽  
Low Molecular Weight Compounds

Herein we present our work on the synthesis, investigation on photophysical properties, interactions with nucleic acids, molecular docking, and imaging application of three carbocyanine dyes. Described low-molecular-weight compounds were found...

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