CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3

Westra, Edze R.; Erp, Paul B. G. van; Künne, Tim; Wong, Shi Pey; Staals, Raymond H.J.; Seegers, Christel L. C.; Bollen, Sander; Jore, Matthijs M.; Semenova, Ekaterina; Severinov, Konstantin; Vos, Willem M. de; Dame, Remus T.; Vries, Renko de; Brouns, Stan J. J.; Oost, John van der

doi:10.1016/j.molcel.2012.03.018

Cited by 487 publications

(668 citation statements)

References 41 publications

Supporting

Mentioning

613

Contrasting

Unclassified

Order By: Relevance

“…The short nucleotide motif adjacent to the protospacer, known as the protospacer-adjacent motif (PAM), is highly conserved among targets of CRISPR/Cas systems (Mojica et al, 2009). Mutations in these nucleotides have been found to disrupt CRISPR-mediated resistance despite complete identity in the protospacer sequence (Semenova et al, 2011(Semenova et al, , 2009Westra et al, 2012).…”

Section: Diversity Of P Acnes Phages In the Human Skinmentioning

confidence: 99%

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

et al. 2015

View full text Add to dashboard Cite

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium-phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey-predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy.

show abstract

Section: Diversity Of P Acnes Phages In the Human Skinmentioning

confidence: 99%

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Cas3-targeting invader DNA colocalizes with Cse1 in the E. coli Cascade complex (30). Additionally, a synthetic E. coli Cas3-Cse1 fusion protein, linked between Cas3 C terminus and Cse1 N terminus, was catalytically active and formed a functional complex with the other four subunits of Cascade (28). Based on these factors, we propose that the Cas3 CTD might be the region responsible for interaction with Cse1.…”

Section: Resultsmentioning

confidence: 95%

“…In Escherichia coli K-12 (type IE), Cascade recognizes and binds crRNA to complimentary sequence in target DNA, generating an RNA mediated displacement loop (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid within double-stranded (ds) target DNA (13). Formation of the R-loop structure induces conformational change in Cascade subunits, triggering recruitment of Cas3 protein that is a nuclease-helicase responsible for degradation of dsDNA (27)(28)(29)(30).…”

mentioning

confidence: 99%

“…Thermus thermophilus HB8 type IE Cas3′′ HD nuclease degrades ssDNA endo-nucleolytically with Mn 2+ or Ni 2+ ions (35). Significantly for understanding CRISPR interference, E. coli type IE Cas3 protein catalyzes Mg 2+ ionand transition metal ion-dependent nicking of the displaced strand of the target dsDNA, after which ATP-dependent unwinding of the target allows for the 3′ to 5′ processive nuclease activity on the nicked ssDNA (28,29,33,35,36). Overall, the data suggests Significance Bacteria can repel invader DNA and RNA molecules by using an adaptive immunity mechanism called clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Gong

Shin

Sun

et al. 2014

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

View full text Add to dashboard Cite

Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HDnucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3′ to 5′ nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3′ to 5′ translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.M any bacteria and archaea can eliminate invading phages and plasmids by activating a defense system that is based on processing of clustered regularly interspaced short palindromic repeats (CRISPRs) by CRISPR-associated (Cas) proteins and various additional proteins. These CRISPR-Cas systems are diverse in structure and function, but common principles of action allow their classification into three major types (I, II, and III) with some further divisions into subtypes (e.g., type IA-F) (1-6).Common CRISPR-mediated immunity processes are usually defined into three stages: (i) acquisition of a short DNA segment (protospacer) from an invading virus or plasmid, and its insertion at the leader-proximal end of a CRISPR locus (7, 8), (ii) generation of small mature CRISPR RNAs (crRNAs) from a longer transcript of a CRISPR locus (9-11), and (iii) interference of foreign nucleic acid invaders by a crRNA-containing ribonucleoprotein effector complex (12-18).Different interference effector complexes characterize the three major CRISPR types. Cascade (Cas complex for antiviral defense) complexes are synonymous with interference in type I CRISPRs, comprising multiple proteins in a helical structure around crRNA that targets invader DNA (13,19, 20). Type II CRISPR systems are characterized by Cas9, a multidomain protein with a bilobed architecture harboring two nuclease sites that together catalyze cleavage of invading DNA (12, 21-24). The Csm system in CRISPR type IIIA and a related Cmr ...

show abstract

“…While we found the motif to be composed of slightly different bases, we believe that these differences likely to arise from the fact that we were able to synthetically control for the presence of the more dominant PAM motif in our sequences, and thus we adopt the previous term AAM. The PAM sequence has been shown to function not only in adaptation but also interference 23 and, given that it lies outside of the acquired spacer, serves as a mechanism of self versus non-self discrimination 14 . Although the AAM lies within the acquired spacer (and thus could promote self-targeting) and additionally lies outside the seed region 24 , it would still be interesting to directly test whether the presence of an AAM similarly influences interference efficiency.…”

mentioning

confidence: 99%

CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria

Shipman

Nivala

Macklis

et al. 2017

Nature

296

220

View full text Add to dashboard Cite

DNA is an excellent medium for data archival. Recent efforts have illustrated the potential for information storage in DNA using synthesized oligonucleotides assembled in vitro1–6. A relatively unexplored avenue of information storage in DNA is the ability to write information into the genome of a living cell by the addition of nucleotides over time. Using the Cas1-Cas2 integrase, the CRISPR-Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity7. Harnessed, this system has the potential to write arbitrary information into the genome8. Here, we use the CRISPR-Cas system to encode images and a short movie into the genomes of a population of living bacteria. In doing so, we push the technical limits of this information storage system and optimize strategies to minimize those limitations. We additionally uncover underlying principles of the CRISPR-Cas adaptation system, including sequence determinants of spacer acquisition relevant for understanding both the basic biology of bacterial adaptation as well as its technological applications. This work demonstrates that this system can capture and stably store practical amounts of real data within the genomes of populations of living cells.

show abstract

CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3

Cited by 487 publications

References 41 publications

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria

Contact Info

Product

Resources

About