Conferring resistance to geminiviruses with the CRISPR-Cas prokaryotic immune system.
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ABSTRACT:
SUBMITTER: Baltes NJ
PROVIDER: S-EPMC8612103 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit - the so-called CRISPR polymerase - which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.
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Project description:CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
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Project description:A stochastic, agent-based mathematical model of the coevolution of the archaeal and bacterial adaptive immunity system, CRISPR-Cas, and lytic viruses shows that CRISPR-Cas immunity can stabilize the virus-host coexistence rather than leading to the extinction of the virus. In the model, CRISPR-Cas immunity does not specifically promote viral diversity, presumably because the selection pressure on each single proto-spacer is too weak. However, the overall virus diversity in the presence of CRISPR-Cas grows due to the increase of the host and, accordingly, the virus population size. Above a threshold value of total viral diversity, which is proportional to the viral mutation rate and population size, the CRISPR-Cas system becomes ineffective and is lost due to the associated fitness cost. Our previous modeling study has suggested that the ubiquity of CRISPR-Cas in hyperthermophiles, which contrasts its comparative low prevalence in mesophiles, is due to lower rates of mutation fixation in thermal habitats. The present findings offer a complementary, simpler perspective on this contrast through the larger population sizes of mesophiles compared to hyperthermophiles, because of which CRISPR-Cas can become ineffective in mesophiles. The efficacy of CRISPR-Cas sharply increases with the number of proto-spacers per viral genome, potentially explaining the low information content of the proto-spacer-associated motif (PAM) that is required for spacer acquisition by CRISPR-Cas because a higher specificity would restrict the number of spacers available to CRISPR-Cas, thus hampering immunity. The very existence of the PAM might reflect the tradeoff between the requirement of diverse spacers for efficient immunity and avoidance of autoimmunity.
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Project description:Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the causal agents of grapevine leafroll disease (GLD), which severely impacts grapevine production in most viticultural regions of the world. The development of virus-resistant plants is a desirable strategy for the efficient control of viral diseases. However, natural resistant resources have not been reported in the genus Vitis, and anti-GLRaV-3 research has been quite limited in grapevine. In this study, by expressing FnCas9 and LshCas13a, we established a highly effective transgenic construct screening system via an optimized Agrobacterium-mediated transient delivery system in grapevine plantlets. Our study indicated that CRISPR/FnCas9 and LshCas13a caused GLRaV-3 inhibition. Moreover, three vectors-pCR01-CP, pCR11-Hsp70h and pCR11-CP-exhibited the most robust inhibition efficiency compared to those targeting other sites and could be further engineered to generate GLRaV-3-resistant grapevine. In addition, the viral interference efficiency of FnCas9 was dependent on its RNA binding activity. The efficiency of virus inhibition was positively correlated with the level of Cas gene expression. Importantly, we demonstrated that LshCas13a had better interference efficiency against viruses than FnCas9. In summary, this study confirmed that these two RNA-targeting CRISPR mechanisms can confer immunity against viruses in grapevine, providing new avenues to control GLRaV-3 or other RNA viruses in fruit crops.
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Project description:A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct 'anti-CRISPR' genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.
| S-EPMC4931913 | biostudies-literature
Project description:CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is a microbial adaptive immune system involved in defense against different types of mobile genetic elements. CRISPR-Cas systems are usually found in bacterial and archaeal chromosomes but have also been reported in bacteriophage genomes and in a few mega-plasmids. Klebsiella pneumoniae is an important member of the Enterobacteriaceae with which they share a huge pool of antibiotic resistance genes, mostly via plasmids. CRISPR-Cas systems have been identified in K. pneumoniae chromosomes, but relatively little is known of CRISPR-Cas in the plasmids resident in this species. In this study, we searched for CRISPR-Cas system in 699 complete plasmid sequences (>50-kb) and 217 complete chromosomal sequences of K. pneumoniae from GenBank and analyzed the CRISPR-Cas systems and CRISPR spacers found in plasmids and chromosomes. We found a putative CRISPR-Cas system in the 44 plasmids from Klebsiella species and GenBank search also identified the identical system in three plasmids from other Enterobacteriaceae, with CRISPR spacers targeting different plasmid and chromosome sequences. 45 of 47 plasmids with putative type IV CRISPR had IncFIB replicon and 36 of them had an additional IncHI1B replicon. All plasmids except two are very large (>200 kb) and half of them carried multiple antibiotic resistance genes including bla CTX-M , bla NDM , bla OXA . To our knowledge, this is the first report of multi drug resistance plasmids from Enterobacteriaceae with their own CRISPR-Cas system and it is possible that the plasmid type IV CRISPR may depend on the chromosomal type I-E CRISPRs for their competence. Both chromosomal and plasmid CRISPRs target a large variety of plasmids from this species, further suggesting key roles in the epidemiology of large plasmids.
| S-EPMC6965323 | biostudies-literature
Project description:Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function.
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Project description:Clustered regularly interspaced short palindromic repeats (CRISPRs) are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21-37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas) protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.
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Project description: Not available
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Project description:CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.
| S-EPMC3651764 | biostudies-literature