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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    • ". This " memory system " can destroy DNA or RNA if reinfection occurs in the same bacteria or in its descendants [14] [15] [16] [17] [18] [19]. Three types of CRISPR loci exist, all of which acquire short pieces of DNA called spacers from foreign DNA elements [20]. "
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    ABSTRACT: Genetic manipulation is a powerful tool to establish the causal relationship between a genetic lesion and a particular pathological phenotype. The rise of CRISPR/Cas9 genome-engineering tools overcame the traditional technical bottleneck for routine site-specific genetic manipulation in cells. To create the perfect in vitro cell model, there is significant interest from the stem cell research community to adopt this fast evolving technology. This review addresses this need directly by providing both the up-to-date biochemical rationale of CRISPR-mediated genome engineering and detailed practical guidelines for the design and execution of CRISPR experiments in cell models. Ultimately, this review will serve as a timely and comprehensive guide for this fast developing technology.
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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.
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    • "These data are strong evidence that A. vinelandii DJ has a functioning CRISPR-Cas system (CASS). It has been proposed that a CASS can work as a defensive mechanism against bacteriophage invasion (Makarova et al., 2006). The A. vinelandii DJ genome has 40 protein-coding genes annotated with the word " phage. "

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