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Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.

ABSTRACT: CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR-Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR-Cas systems.

SUBMITTER: Vorontsova D

PROVIDER: S-EPMC4678832 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

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Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.

Vorontsova Daria D Datsenko Kirill A KA Medvedeva Sofia S Bondy-Denomy Joseph J Savitskaya Ekaterina E EE Pougach Ksenia K Logacheva Maria M Wiedenheft Blake B Davidson Alan R AR Severinov Konstantin K Semenova Ekaterina E

Nucleic acids research 20151119 22

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target ...[more]

PMID: 26586803

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Project description:Antibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this "CRISPR tolerance." We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.IMPORTANCE CRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in E. faecalis with negligible off-target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR-induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.

Project description:BackgroundCRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism involving the assembly of small crRNA molecules and Cas proteins into a ribonucleoprotein (RNP) effector complex, and formation of an R-loop structure upon binding to the target DNA. Extensive research on the I-E subtype established the prototypical mechanism of DNA interference in type I systems, where the coordinated action of a ribonucleoprotein Cascade complex and Cas3 protein destroys foreign DNA. However, diverse protein composition between type I subtypes suggests differences in the mechanism of DNA interference that could be exploited for novel practical applications that call for further exploration of these systems.ResultsHere we examined the mechanism of DNA interference provided by the type I-F1 system from Aggregatibacter actinomycetemcomitans D7S-1 (Aa). We show that functional Aa-Cascade complexes can be assembled not only with WT spacer of 32 nt but also with shorter or longer (14-176 nt) spacers. All complexes guided by the spacer bind to the target DNA sequence (protospacer) forming an R-loop when a C or CT protospacer adjacent motif (PAM) is present immediately upstream the protospacer (at -1 or -2,-1 position, respectively). The range of spacer and protospacer complementarity predetermine the length of the R-loop; however, only R-loops of WT length or longer trigger the nuclease/helicase Cas2/3, which initiates ATP-dependent unidirectional degradation at the PAM-distal end of the WT R-loop. Meanwhile, truncation of the WT R-loop at the PAM-distal end abolishes Cas2/3 cleavage.ConclusionsWe provide a comprehensive characterisation of the DNA interference mechanism in the type I-F1 CRISPR-Cas system, which is different from the type I-E in a few aspects. First, DNA cleavage initiation, which usually happens at the PAM-proximal end in type I-E, is shifted to the PAM-distal end of WT R-loop in the type I-F1. Second, the R-loop length controls on/off switch of DNA interference in the type I-F1, while cleavage initiation is less restricted in the type I-E. These results indicate that DNA interference in type I-F1 systems is governed through a checkpoint provided by the Cascade complex, which verifies the appropriate length for the R-loop.

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Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.

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Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.

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