Transcription, processing and function of CRISPR cassettes in <i>Escherichia coli</i>

Pougach, Ksenia; Semenova, Ekaterina; Bogdanova, Ekaterina; Datsenko, Kirill A.; Djordjevic, M.; Wanner, Barry L.; Severinov, Konstantin

doi:10.1111/j.1365-2958.2010.07265.x

Cited by 211 publications

(249 citation statements)

References 31 publications

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“…46 In E. coli, crRNA abundance increased with elevated levels of CasE. 23,39,46 In agreement, we observe a similar increase in crRNA levels for the Δcas strain overexpressing Csy4 compared with the wt (compare Fig. 2C and B).…”

Section: Methodssupporting

confidence: 81%

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Csy4 is responsible for CRISPR RNA processing inPectobacterium atrosepticum

Przybilski

Richter²,

Gristwood³

et al. 2011

RNA Biology

100

118

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Section: Methodssupporting

confidence: 81%

“…It is yet to be determined what regulates CRISPR/Cas expression in P. atrosepticum. H-NS and LeuO differentially regulate CRISPR/Cas activity in E. coli, 39,46,47 and therefore may also have a role in P. atrosepticum.…”

Section: Methodsmentioning

confidence: 99%

Csy4 is responsible for CRISPR RNA processing inPectobacterium atrosepticum

Przybilski

Richter²,

Gristwood³

et al. 2011

RNA Biology

100

118

View full text Add to dashboard Cite

“…Upon induction, the level of g8 crRNA was the same in both strains (Fig. S1), an expected result, as the crRNA precursor is transcribed from its own promoter (31). Spacers were efficiently acquired by exponentially growing KD456 cultures transformed with either plasmid (Fig.…”

Section: Increased Cas1 and Cas2 Production Leads To Efficient Primedsupporting

confidence: 53%

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

Semenova

Savitskaya

Musharova

et al. 2016

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

Self Cite

111

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Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acquisition of spacers-short fragments of foreign DNA. For the type I-E CRISPR-Cas system from Escherichia coli, efficient "primed" adaptation requires Cas effector proteins and a CRISPR RNA (crRNA) whose spacer partially matches a segment (protospacer) in target DNA. Primed adaptation leads to selective acquisition of additional spacers from DNA molecules recognized by the effector-crRNA complex. When the crRNA spacer fully matches a protospacer, CRISPR interference-that is, target destruction without acquisition of additional spacers-is observed. We show here that when the rate of degradation of DNA with fully and partially matching crRNA targets is made equal, fully matching protospacers stimulate primed adaptation much more efficiently than partially matching ones. The result indicates that different functional outcomes of CRISPR-Cas response to two kinds of protospacers are not caused by different structures formed by the effector-crRNA complex but are due to the more rapid destruction of targets with fully matching protospacers.CRISPR-Cas | CRISPR interference | primed CRISPR adaptation

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“…In contrast, we found that cas6, cas10, and csm4 are required for the accumulation of crRNAs in vivo. It has been observed that unprocessed crRNA precursors are degraded by cellular nucleases (29), and therefore, we interpret the absence of primer extension products and Northern blot signals in these mutants as lack of primary processing of the precursor. The Cas6 homolog from P. furiosus cleaves synthetic RNA molecules containing repeat sequences (8,24) and most likely, performs the endoribonucleolytic cleavage of the crRNA precursor in S. epidermidis.…”

Section: Discussionmentioning

confidence: 82%

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.

181

205

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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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Transcription, processing and function of CRISPR cassettes in Escherichia coli

Cited by 211 publications

References 31 publications

Csy4 is responsible for CRISPR RNA processing inPectobacterium atrosepticum

Csy4 is responsible for CRISPR RNA processing inPectobacterium atrosepticum

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

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

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