Makarova, K.S. , Grishin, N.V. , Shabalina, S.A. , Wolf, Y.I. & Koonin, E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 1, 7

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
Biology Direct (Impact Factor: 4.66). 02/2006; 1(1):7. DOI: 10.1186/1745-6150-1-7
Source: PubMed


All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis.
The protein sequences of the numerous cas gene products were classified into approximately 25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and/or that the dominant phages and plasmids turn over rapidly.
We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the cas genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids.

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    • "[8] Recently, these palindrome repeats and the cas-genes, associated with them, have been found to function as a new defence mechanism in prokaryotic cells against invading phages and plasmids.[9] [10] The regular repeats are interspaced with short sequences named 'spacers' which derive from foreign genetic elements.[9] [11] [12] When, for example, the bacterial host is under a bacteriophage attack, it acquires a new spacer sequence within its CRISPR locus that matches a DNA sequence in the phage genome, referred to as a 'protospacer'.[13] "
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    ABSTRACT: The function of several families of clustered regularly interspaced palindrome repeats (CRISPRs) in prokaryotic genomes was recently found to be related to the protection of bacterial cells against the expression of foreign DNA, originating from plasmids or bacteriophages. The present study was the first attempt to screen a broader number of Lactobacillus delbrueckii ssp. bulgaricus strains, widely used in yoghurt and cheese production, for the presence of CRISPRs. Database search of four completely sequenced L. delbrueckii ssp. bulgaricus genomes indicated the presence of CRISPR2 in three of them - ATCC 11842, ATCC BAA-365 and ND02, and the presence of CRISPR3 in strain 2038. In the first three strains, the CRISPR2 was invariably located between a 3′-5′ exonuclease gene and a gene for a ppGpp-synthetase. The location of CRISPR3 in strain 2038 was between a histidine-kinase gene and an acetyl-CoA acetyltransferase gene, 2 kbp downstream of the CRISPR2 locus in ATCC 11842. Specific primers were designed to amplify with polymerase chain reaction the target regions containing the potential CRISPR2 and / or CRISPR3 in a total of 33 L. delbrueckii ssp. bulgaricus strains. Thirteen strains yielded a high molecular mass product corresponding in size and location to CRISPR2 of the type strain ATCC 11842, while another 17 strains indicated the presence of potential CRISPR3, analogous to that of strain 2038. Three strains did not indicate the presence of CRISPRs. Interestingly, none of the tested strains carried both CRISPR2 and CRISPR3 simultaneously in its genome at the investigated region.
    Biotechnology & Biotechnological Equipment 02/2015; 29(3):1-6. DOI:10.1080/13102818.2015.1013351 · 0.30 Impact Factor
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    • "Type II clustered regularly interspaced short palindromic repeats and their associated systems (CRISPR/Cas9 systems) from Streptococcus pyogenes are the most widely used CRISPR/Cas9 genomic engineering platform to date. Target site recognition relies on the Cas9-mediated Watson-Crick base pairing between a short stretch of CRISPR repeat RNA (originally named as 'spacer') and one strand of target DNA (known as the 'protospacer' sequence) (Jinek et al., 2012; Makarova et al., 2006). This protospacer sequence must be immediately followed by a 'NGG' (or 'NAG' with less efficiency) tri-nucleotide protospacer adjacent motif (PAM) on the opposite strand (Mojica et al., 2009); the presence of PAM is crucial for Cas9 target recognition. "
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    ABSTRACT: Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. In this Review, we summarize the common approaches and applications of these still-evolving tools as they are being used in the most popular model developmental systems. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms. KEY WORDS: Genome engineering, Transcription activator-like effector nuclease (TALEN), Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas9), Zinc finger nuclease (ZFN), Model organisms
    Development 11/2014; 141(21). DOI:10.1242/dev.102186 · 6.46 Impact Factor
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    • ". Next, we took the results and searched them against protein family databases CDD (Makarova et al., 2011a), COG (Makarova et al., 2006) and Pfam (Punta et al., 2012) using RPS-Blast (Marchler-Bauer et al., 2011). Then, we generated new models and supermodels from those databases. "
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    ABSTRACT: Motivation: The discovery of CRISPR-Cas systems almost 20 years ago rapidly changed our perception of the bacterial and archaeal immune systems. CRISPR loci consist of several repetitive DNA sequences called repeats, inter-spaced by stretches of variable length sequences called spacers. This CRISPR array is transcribed and processed into multiple mature RNA species (crRNAs). A single crRNA is integrated into an interference complex, together with CRISPR-associated (Cas) proteins, to bind and degrade invading nucleic acids. Although existing bioinformatics tools can recognize CRISPR loci by their characteristic repeat-spacer architecture, they generally output CRISPR arrays of ambiguous orientation and thus do not determine the strand from which crRNAs are processed. Knowledge of the correct orientation is crucial for many tasks, including the classification of CRISPR conservation, the detection of leader regions, the identification of target sites (protospacers) on invading genetic elements and the characterization of protospacer-adjacent motifs. Results: We present a fast and accurate tool to determine the crRNA-encoding strand at CRISPR loci by predicting the correct orientation of repeats based on an advanced machine learning approach. Both the repeat sequence and mutation information were encoded and processed by an efficient graph kernel to learn higher-order correlations. The model was trained and tested on curated data comprising >4500 CRISPRs and yielded a remarkable performance of 0.95 AUC ROC (area under the curve of the receiver operator characteristic). In addition, we show that accurate orientation information greatly improved detection of conserved repeat sequence families and structure motifs. We integrated CRISPRstrand predictions into our CRISPRmap web server of CRISPR conservation and updated the latter to version 2.0. Availability: CRISPRmap and CRISPRstrand are available at Supplementary information: Supplementary data are available at Bioinformatics online.
    Bioinformatics 09/2014; 30(17):i489-i496. DOI:10.1093/bioinformatics/btu459 · 4.98 Impact Factor
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