Different genome stability proteins underpin primed and naïve adaptation in<i>E. coli</i>CRISPR-Cas immunity

Ivančić-Baće, Ivana; Cass, Simon; Wearne, Stephen; Bolt, Edward L.

doi:10.1093/nar/gkv1213

Cited by 88 publications

(106 citation statements)

References 75 publications

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“…The adaptation proteins Cas1 and Cas2 are conserved components of CRISPR systems, and these two proteins have been shown to be necessary for new spacer acquisition in the type I-E systems (7,21,55). In E. coli, Cas1 and Cas2 assemble into a stable integration complex, and a structure of this complex fits with high confidence into a portion of the electron density in our Cas1-2/3 reconstruction (Fig.…”

Section: Discussionmentioning

confidence: 58%

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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Section: Discussionmentioning

confidence: 58%

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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“…Recent work has demonstrated the involvement of both the RecBCD complex and the RecG protein in naive and primed CRISPR adaptation, respectively (28,32). The RecBCD complex is required for naive adaptation in the type I-E system of E. coli and likely contributes to adaptation through single-stranded DNA generation at double-stranded breaks, such as those encountered during replication, thereby providing a substrate for Cas1 (28).…”

Section: Methodsmentioning

confidence: 99%

“…The RecBCD complex is required for naive adaptation in the type I-E system of E. coli and likely contributes to adaptation through single-stranded DNA generation at double-stranded breaks, such as those encountered during replication, thereby providing a substrate for Cas1 (28). Regarding primed adaptation, in the E. coli type I-E system, RecG and PriA were shown to be required for primed adaptation, presumably through R-loop removal after crRNA binding, allowing the Cas1-Cas2 complex access to the single-stranded DNA generated by Cas3 (32). We tested if these proteins were playing a similar role in the type I-F system of P. aeruginosa using the biofilm enrichment system and hypothesized that since all spacer acquisition in our system is likely primed, deletion of the recD gene would not impact spacer acquisition, while deletion of the recG gene would impact new spacer insertion.…”

Section: Methodsmentioning

confidence: 99%

Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay

et al. 2016

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CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer. IMPORTANCEOur understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of Escherichia coli, or cas1-cas2 overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows P. aeruginosa to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system of P. aeruginosa. Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that many P. aeruginosa strains maintain a CRISPR-Cas system.

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“…According to one proposed model [69], replication forks in the invader's DNA are blocked by the Cascade complex bound to the priming crRNA, enabling the RecG helicase and the Cas3 helicase/nuclease proteins to attack the DNA. The ends at the collapsed forks then could be targeted by RecBCD which provides DNA fragments for new spacer generation [69].…”

Section: Crispr Adaptationmentioning

confidence: 99%

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

Mohanraju

Makarova

Zetsche

et al. 2016

Science

572

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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Different genome stability proteins underpin primed and naïve adaptation inE. coliCRISPR-Cas immunity

Cited by 88 publications

References 75 publications

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

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

Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay

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

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