CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array

Wei, Yunzhou; Terns, Michael P.

doi:10.1016/j.molcel.2016.06.004

Cited by 5 publications

(3 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the type I CRISPR-Cas system, binding of IHF to the leader creates the required DNA topology for spacer integration at the 5′-end of the array. Given the absence of IHF homologs in Gram-positive bacteria (including the host used in our studies, S. aureus ) it is possible that there are other factors that perform a similar function for type II CRISPR-Cas systems (Wei and Terns, 2016). Alternatively, the type II Cas1–Cas2 complex could be sufficient to catalyze polarized spacer integration without a requirement for additional host factors (Rollie et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration

2016

View full text Add to dashboard Cite

SUMMARY CRISPR-Cas systems defend prokaryotes against viruses and plasmids. Short DNA segments of the invader, known as spacers, are stored in the CRISPR array as immunological memories. New spacers are added invariably to the 5′ end of the array, therefore the first spacer matches the latest foreign threat. Whether this highly polarized order of spacer insertion influences CRISPR-Cas immunity has not been explored. Here we show that a conserved sequence located immediately upstream of the CRISPR array specifies the site of new spacer integration. Mutation of this sequence results in erroneous incorporation of new spacers into the middle of the array. We show that spacers added through polarized acquisition give rise to more robust CRISPR-Cas immunity than spacers added to the middle of the array. This study demonstrates that the CRISPR-Cas system specifies the site of spacer integration to optimize the immune response against the most immediate threat to the host.

show abstract

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration

2016

View full text Add to dashboard Cite

show abstract

“…Interestingly, wherever IBS is conserved in type I-E systems, we also noted a strong correlation for the existence of IAS suggesting that these two sites co-evolve to preserve the CRISPR adaptation active (Figure 3A). However, since few organisms that harbour type I-E system in our analysis lack IHF (27%), it is possible to envisage the participation of other DNA architectural proteins such as HU or other auxiliary Cas proteins to facilitate protospacer integration (62,63). It may be noted that HU is structurally similar to IHF; however, unlike IHF, it binds DNA non-specifically.…”

Section: Discussionmentioning

confidence: 99%

Asymmetric positioning of Cas1–2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system

Yoganand¹,

Sivathanu²,

Nimkar³

et al. 2016

Nucleic Acids Res

View full text Add to dashboard Cite

CRISPR–Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli. Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1–2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1–2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1–2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion.

show abstract

“…IHF also stimulates leader-proximal integration catalyzed by Type I-F Cas1-Cas2-3 integrase of Pectobacterium atrosepticum (79). In contrast, IHF is absent in numerous prokaryotes that contain active CRISPR-Cas systems, including Gram-positive bacteria and archaea (80). With Sulfolobus solfataricus, crude extract addition was shown to stimulate in vitro site-specific integration of a protospacer in the Type I-A CRISPR system of this archaea (21).…”

Section: Discussionmentioning

confidence: 97%

Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1–Cas2 integrase

Plateau

Moch

Blanquet

2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

Edited by Patrick SungSite-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro. However, this nonspecific integration activity is rare in vivo. Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mM spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.

show abstract

CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array

Abstract: In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA.

Cited by 5 publications

References 11 publications

CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration

CRISPR-Cas Systems Optimize Their Immune Response by Specifying the Site of Spacer Integration

Asymmetric positioning of Cas1–2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system

Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1–Cas2 integrase

Contact Info

Product

Resources

About