Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system

Arslan, Zihni; Hermanns, Veronica; Wurm, Reinhild; Wagner, Rolf; Pul, Ümit

doi:10.1093/nar/gku510

Cited by 125 publications

(138 citation statements)

References 48 publications

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“…Although this step may be non-essential, it probably enhances the efficiency of the overall process and its specificity toward invading DNA. (2) Selection of DNA fragments for (proto)spacers by scanning for potential PAMs (after partial target unwinding) by one of the four Cas1 subunits of the Cas1-Cas2 complex [63]. (3) Measuring of the selected protospacer generating fragments of the correct size with 3' hydroxyl groups by Cas1 nuclease.…”

Section: Crispr Adaptationmentioning

confidence: 99%

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

560

460

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Background: Prokaryotes have evolved multiple systems to combat invaders such as viruses and plasmids. Examples of such defence systems include receptor masking, restriction-modification (R-M systems), DNA interference (Argonaute), bacteriophage exclusion (BREX or PGL) and abortive infection, all of which act in an innate, non-specific manner. In addition, prokaryotes have evolved adaptive, heritable immune systems, i.e. clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated proteins (CRISPR-Cas). Adaptive immunity is conferred by the integration of DNA sequences from an invading element into the CRISPR array (adaptation), which is transcribed into long pre-CRISPR (pre-cr) RNAs and processed into short crRNAs (expression), which guide Cas proteins to specifically degrade the cognate DNA on subsequent exposures (interference). Advances:A plethora of distinct CRISPR-Cas systems are represented in genomes of most archaea and almost half of the bacteria. The latest CRISPR-Cas classification scheme delineates two classes that are each subdivided into three types. Integration of biochemistry and molecular genetics has contributed significantly to revealing many of the unique features of the variant CRISPR-Cas types. Additionally, structural analysis and single molecule studies have further advanced our understanding of the molecular basis of CRISPR-Cas functionality. Recent progress includes relevant steps in the adaptation stage, when fragments of foreign DNA are processed and incorporated as new spacers into the CRISPR array. In addition, three novel CRISPR-Cas types (IV, V, and VI) have been identified, and in particular, the type V interference complexes have been experimentally characterized. Moreover, the ability to easily program sequence-specific DNA targeting and cleavage by CRISPRCas components, as demonstrated for Cas9 and Cpf1, allows for the application of CRISPR-Cas components as highly effective tools for genetic engineering and gene regulation in a wide range of eukaryotes and prokaryotes.The pressing issue of off-target cleavage by the Cas9 nuclease is being actively addressed using structure-guided engineering.Outlook: Although our understanding of the CRISPR-Cas system has increased tremendously over the past few years, much remains to be done. About the evolution of CRISPR-Cas systems, the continuing discovery of novel CRISPR-Cas variants will provide direct tests of the recently proposed modular scenario. The recent discovery and characterization of new CRISPR-Cas types with many unique features implies that our current knowledge has relatively limited power for predicting the functional details of distantly related variants. Hence, newly discovered CRISPR-Cas systems need to be thoroughly dissected with the aforementioned multi-disciplinary approaches to gain insight in their biological role, to unravel their molecular mechanism, and to harness their potential for biotechnology. As to the biology, key outstanding questions include the ecological roles of microbial ...

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Section: Crispr Adaptationmentioning

confidence: 99%

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

560

460

View full text Add to dashboard Cite

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“…1A) (7,(18)(19)(20). Cas2 proteins play a critical role in CRISPR adaptation (i.e., the integration of foreign DNA into CRISPR loci) (21)(22)(23)(24)(25), and fusion of the Cas2 adaptation protein to the Cas3 interference protein suggests a functional connection between the two stages of CRISPR immunity. In fact, recent work by Vorontsova et al has shown that new sequence acquisition (both naïve and primed) in type I-F systems requires all of the Cas proteins, including those that were previously thought to be involved exclusively in interference (26).…”

mentioning

confidence: 99%

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

Rollins

Chowdhury

Carter

et al. 2017

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

102

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Significance Prokaryotes have adaptive immune systems that rely on CRISPRs (clustered regularly interspaced short palindromic repeats) and diverse CRISPR-associated ( cas ) genes. Cas1 and Cas2 are conserved components of CRISPR systems that are essential for integrating fragments of foreign DNA into CRISPR loci. In type I-F immune systems, the Cas2 adaptation protein is fused to the Cas3 interference protein. Here we show that the Cas2/3 fusion protein from Pseudomonas aeruginosa stably associates with the Cas1 adaptation protein, forming a 375-kDa propeller-shaped Cas1–2/3 complex. We show that Cas1, in addition to being an essential adaptation protein, also functions as a repressor of Cas2/3 nuclease activity and that foreign DNA binding by the CRISPR RNA-guided surveillance complex activates the Cas2/3 nuclease.

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“…The acquisition module appropriates spacers from foreign DNA into CRISPR arrays and consists of proteins Cas1 and Cas2, homologous in all CRISPR-Cas systems (4). The Cas1 and Cas2 proteins from Escherichia coli alone are able to perform the spacer acquisition reaction in vitro (5) and are also sufficient for spacer acquisition in vivo in the absence of other Cas proteins (6,7). Whenever a new spacer is acquired, a new copy of CRISPR repeat is also generated (1,6).…”

mentioning

confidence: 99%

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.

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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Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system

Cited by 125 publications

References 48 publications

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity

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

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