Self versus non-self discrimination during CRISPR RNA-directed immunity

Marraffini, Luciano A.; Sontheimer, Erik J.

doi:10.1038/nature08703

Cited by 587 publications

(538 citation statements)

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“…The average number of events for self-targeting spacer types was 1.01 with a spacer frequency of 1.36, indicating that autoimmunity generates unstable and likely non-viable genotypes. This reflects an imperfect ability of the host to differentiate self from nonself 23 and indicates strong selection against cells that acquire self-targeting spacers, presumably because self-targeting spacers are lethal.…”

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confidence: 99%

Strong bias in the bacterial CRISPR elements that confer immunity to phage

et al. 2013

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal. In experiments that recover over half a million spacers, we observe early dominance by a few spacer sub-populations and rapid oscillations in sub-population abundances. In two CRISPR systems and in replicate experiments, a few spacers account for the majority of spacer sequences. Nearly all phage locations targeted by the acquired spacers have a proto-spacer adjacent motif (PAM), indicating PAMs are involved in spacer acquisition. We detect a strong and reproducible bias in the phage genome locations from which spacers derive. This may reflect selection for specific spacers based on location and effectiveness.

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confidence: 99%

Strong bias in the bacterial CRISPR elements that confer immunity to phage

et al. 2013

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“…Cleavage occurs 8 nt upstream of the beginning of the spacer sequence, leaving a 5′ handle (6) or crRNA tag (18) on the 5′ end. In Type III systems, crRNA intermediates are subject to additional nucleolytic attack at the 3′ end, generating mature crRNAs with reduced length (18,19). This final maturation step seems to be absent in Type I systems (10,12,13,15).…”

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confidence: 99%

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site

Hatoum-Aslan

Maniv

Marraffini

2011

Proc. Natl. Acad. Sci. U.S.A.

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Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.conjugation | genetic interference | antisense RNA C lustered, regularly interspaced, short palindromic repeat (CRISPR) sequences are present in ∼40% of eubacterial genomes and nearly all archaeal genomes sequenced to date. CRISPR loci consist of short (∼24-48 nt) repeats separated by similarly sized unique spacers (1-4). CRISPR systems protect against bacteriophage and plasmid infection by a genetic interference mechanism that relies on the identity between CRISPR spacers and the invading targets (5-7). CRISPR arrays are transcribed into a long precursor containing spacers and repeats that are processed into small CRISPR RNAs (crRNAs) by dedicated CRISPR-associated (Cas) endoribonucleases (6,8,9). crRNAs act as guides for a targeting complex (10-15) that cleaves the genetic material of the invading bacteriophage or plasmid (16).In many prokaryotes, the biogenesis of mature crRNAs can be divided into two stages: (i) a primary cleavage of the crRNA precursor within repeat sequences that generates intermediate crRNAs containing a full spacer flanked by partial repeats, and (ii) a final maturation event where intermediate crRNAs are subject to additional nucleolytic digestion at one end. Repeat spacer arrays and their adjacent cas genes are classified into three CRISPR/Cas types (I-III) (17) that undergo different mechanisms of crRNA processing. In Types I and III CRISPR/Cas systems, primary processing is achieved by Cas6 endoribonucleases and results in crRNA intermediates with 5′-hydroxyl and 3′-phosphate or 2′-3′-cyclic phosphate ends (9,10,12,13). Cleavage occurs 8 nt upstream of the beginning of the spacer sequence, leaving a 5′ handle (6) or crRNA tag (18) on the 5′ end. In Type III systems, crRNA intermediates are subject to additional nucleolytic attack at the 3′ end, generating mature crRNAs with reduced...

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“…In these systems, the 5' handle of a crRNA interacts with a repeat sequence in the CRISPR locus. This interaction probably prevents nuclease recruitment, thereby preventing cleavage of self DNA (Marraffini and Sontheimer, 2010).…”

Section: Target Recognition and Degradationmentioning

confidence: 99%

“…Cascade Thermofilum pendens (Hrle et al, 2014) I-E Cse1, repeat group 2 Cas6 homologues Escherichia coli (Westra et al, 2010) II-C lack of Csn2 and Cas4 Neisseria meningitides III-A Csm2 Cas6 homologues, Csm Staphylococcus epidermidis (Marraffini and III Cas10 Csm Sontheimer, 2008) III-B Cmr5 Cas6 homologues, Cmr Thermus thermophilus (Staals et al, 2013), Cmr RNA Sulfolubus solfataricus (Zhang et al, 2012b) is PAM-independent and is based on the proximity of a repeat sequence to a spacer sequence in the CRISPR locus, which hinders its cleavage (Marraffini and Sontheimer, 2010). The current classification of CRISPR-Cas systems is based on the sequences of the cas genes, the sequences of the repeats within the CRISPR arrays, and the organization of the cas operons (Makarova et al, 2011).…”

Section: Structure Of the Crispr-cas Systemmentioning

confidence: 99%

“…In most cases, the CRISPR-Cas system deals with this problem by utilizing protospacer adjacent motifs (PAMs) (Sashital et al, 2012),which are present in the target DNA but not the CRISPR array. DNA cleavage occurs only if the correct PAM sequence is present (Jinek et al, 2012; Semenova et al, 2011;Westra et al, 2013) is PAM-independent and is based on the proximity of a repeat sequence to a spacer sequence in the CRISPR locus, which hinders its cleavage (Marraffini and Sontheimer, 2010). The current classification of CRISPR-Cas systems is based on the sequences of the cas genes, the sequences of the repeats within the CRISPR arrays, and the organization of the cas operons (Makarova et al, 2011).…”

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confidence: 99%

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CRISPR-Cas Systems in Prokaryotes

Burmistrz

Pyrć

2015

Pol J Microbiol

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A b s t r a c tProkaryotic organisms possess numerous strategies that enable survival in hostile conditions. Among others, these conditions include the invasion of foreign nucleic acids such as bacteriophages and plasmids. The clustered regularly interspaced palindromic repeats-CRISPRassociated proteins (CRISPR-Cas) system provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat. This mechanism of immunity is based on short fragments of foreign DNA incorporated within the hosts genome. After transcription, these fragments guide protein complexes that target foreign nucleic acids and promote their degradation. The aim of this review is to summarize the current status of CRISPR-Cas research, including the mechanisms of action, the classification of different types and subtypes of these systems, and the development of new CRISPR-Cas-based molecular biology tools. K e y w o r d s: CRISPR-Cas, prokaryotesBurmistrz M. and Pyrć K. 3 194 spacer sequences can be of either foreign or self-origin (Stern et al., 2010;Vercoe et al., 2013). The proteomic component is responsible for the incorporation of new template sequences, processing them into a form that enables base pairing with target nucleic acids, as well as for scanning and cleavage of target DNA or RNA. Of note, not all CRISPR-Cas systems discovered to date are active (Haft et al., 2005;van der Ploeg, 2009). Structure of the CRISPR-Cas systemThe genomic component of the CRISPR-Cas system is formed by a series of variable spacers, which in some cases share sequence similarity with viruses, plasmids, or bacteria. These regions are interspaced with repeat sequences that are identical or almost identical within a single CRISPR cassette. The length of the repeat sequences varies between 25 and 40 nt, whereas the length of the spacer sequences varies between 21 and 72 nt. As mentioned above, some spacers show high homology with foreign nucleic acids, but the origin of a significant percentage of spacers remains unknown. The sequence homology between the spacer and the target is the major determinant of nucleic acid degradation of the target. Some bacterial species contain more than one CRISPR locus within their genome (Louwen et al., 2014). Depending on the specific bacterial species or strain, a CRISPR locus may contain from a few to several hundred repeat spacer units; however, most commonly, a single CRISPR locus contains approximately 50 units. The CRISPR repeat sequences play an important role during both the acquisition of new spacers and the transcription and maturation of CRISPR RNA (crRNA). Based on the sequence similarity, the CRISPR repeats are assigned into groups (Kunin et al., 2007). These groups were taken into account in the current classification of CRISPR-Cas systems (Makarova et al., 2011). Although most CRISPR arrays are located on chromosomal DNA, there are examples of CRISPRs located on plasmids (Godde and Bickerton, 2006). In general, CRISPR loci are flanked by A/T rich leader sequences containing...

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Self versus non-self discrimination during CRISPR RNA-directed immunity

Cited by 587 publications

References 34 publications

Strong bias in the bacterial CRISPR elements that confer immunity to phage

Strong bias in the bacterial CRISPR elements that confer immunity to phage

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site

CRISPR-Cas Systems in Prokaryotes

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