CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of<i><i>Escherichia coli</i></i>

Díez-Villaseñor, César; Guzmán, Noemí M.; Almendros, Cristóbal; Garcı́a-Martı́nez, Jesús; Mojica, Francisco J. M.

doi:10.4161/rna.24023

Cited by 125 publications

(155 citation statements)

References 58 publications

(118 reference statements)

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“…Similarly, Streptococcus thermophilus with a catalytically inactive Cas9 resulted in a major increase of spacers derived from the host genome [52]. In addition, there is a strong preference for the integration of plasmid over chromosomal spacer sequences [51,53,54] with plasmid sequences incorporated 100-1000 times more frequently than host DNA [55]. Spacer acquisition in E. coli requires active replication of the protospacer-containing DNA [55].…”

Section: Crispr Adaptationmentioning

confidence: 99%

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

Mohanraju

Makarova

Zetsche

et al. 2016

Science

571

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

571

460

View full text Add to dashboard Cite

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“…Selection of invader DNA fragments (protospacers) by the adaptation machinery requires the presence of a short (3-to 7-base-pair An important goal toward understanding CRISPR adaptation is identifying the proteins (Cas and non-Cas) responsible for novel spacer acquisition in CRISPR loci in diverse CRISPR-Cas systems. Genetic studies indicate that overexpression of Cas1 and Cas2-the only Cas proteins universal to all CRISPR-Cas systems-is sufficient to induce adaptation in the absence of other Cas proteins in Type I systems such as that found in Escherichia coli (Datsenko et al 2012;Yosef et al 2012;Diez-Villasenor et al 2013;Nunez et al 2014).Limited information is available regarding the transacting factors required for adaptation in Type II CRISPRCas systems (and no work has yet been done regarding adaptation in Type III systems). The three distinct Type II subtypes (Chylinski et al 2014) each contain (1) three Cas proteins (Cas1, Cas2, and Cas9), (2) a trans-activating crRNA (tracrRNA) (Deltcheva et al 2011), and (3) the CRISPR array (Fig.…”

mentioning

confidence: 99%

“…Interestingly, a fourth Cas protein is found in Type II-A (Csn2) and Type II-B (Cas4) systems but not Type II-C systems (Chylinski et al 2014). Cas1 and Cas2 are presumed to be essential for adaptation in Type II systems, given their vital role in adaptation in Type I systems (Datsenko et al 2012;Yosef et al 2012;Diez-Villasenor et al 2013;Nunez et al 2014). Genetic studies (cas gene disruptions) in Streptococcus thermophilus suggest a specific requirement for Csn2 in Type II-A adaptation (Barrangou et al 2007).…”

mentioning

confidence: 99%

Cas9 function and host genome sampling in Type II-A CRISPR–Cas adaptation

Wei¹,

Terns²,

2015

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To acquire the ability to recognize and destroy virus and plasmid invaders, prokaryotic CRISPR-Cas systems capture fragments of DNA within the host CRISPR locus. Our results indicate that the process of adaptation by a Type II-A CRISPR-Cas system in Streptococcus thermophilus requires Cas1, Cas2, and Csn2. Surprisingly, we found that Cas9, previously identified as the nuclease responsible for ultimate invader destruction, is also essential for adaptation. Cas9 nuclease activity is dispensable for adaptation. In addition, our studies revealed extensive, unbiased acquisition of the selftargeting host genome sequence by the CRISPR-Cas system that is masked in the presence of active target destruction.Supplemental material is available for this article.Received December 17, 2014; revised version accepted January 15, 2015.CRISPR-Cas systems provide prokaryotes with adaptive immunity against invaders such as viruses/phages and plasmids (Terns and Terns 2011;Barrangou and Marraffini 2014;Heler et al. 2014;van der Oost et al. 2014). CRISPR-Cas systems target invaders using information stored in CRISPRs: loci that contain alternating units of an identical repeat (repeats) and short invader-derived sequences (spacers) (Fig. 1A). CRISPR transcripts are processed to a battery of CRISPR RNAs (crRNAs) that each contains a unique invader guide sequence (and common repeat sequence). The crRNAs associate with Cas proteins to form effector complexes that recognize and degrade invading nucleic acids to effect immunity (Terns and Terns 2011;Barrangou and Marraffini 2014;Heler et al. 2014;van der Oost et al. 2014). Diverse CRISPR-Cas systems are prevalent in bacteria and archaea and are categorized into three compositionally distinct groups (Types I-III), with multiple subtypes within each group (Haft et al. 2005;Makarova et al. 2011).The initial step of capturing short fragments of invasive DNA into CRISPR loci (''adaptation'' or ''spacer acquisition'') is the least understood aspect of the CRISPR immune pathway. Adaptation appears to be a rare event but generates subpopulations of organisms that can survive infection. It has been proposed that the mechanism involves identification of ''foreign'' sequences for incorporation into the CRISPR (Datsenko et al. 2012;Yosef et al. 2012;Diez-Villasenor et al. 2013;Nunez et al. 2014), although host genome sequences have also been observed in CRISPRs at very low frequencies (Stern et al. 2010;Jiang et al. 2013;Paez-Espino et al. 2013). Selection of invader DNA fragments (protospacers) by the adaptation machinery requires the presence of a short (3-to 7-base-pair An important goal toward understanding CRISPR adaptation is identifying the proteins (Cas and non-Cas) responsible for novel spacer acquisition in CRISPR loci in diverse CRISPR-Cas systems. Genetic studies indicate that overexpression of Cas1 and Cas2-the only Cas proteins universal to all CRISPR-Cas systems-is sufficient to induce adaptation in the absence of other Cas proteins in Type I systems such as that found in Escherichia col...

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“…However, these mechanisms would not prevent cell death if a spacer is acquired that targets the host chromosome, and accordingly, evidence from the type I-E system of Escherichia coli suggests that the bacteria have evolved to preferentially sample foreign DNA over chromosomal DNA during naive CRISPR adaptation. It was observed previously in CRISPR adaptation studies using E. coli as a model system that the cell is roughly 100 to 1,000 times more likely to incorporate plasmid DNA over chromosomal DNA into its CRISPRs after normalizing for their respective size (27,28). Recent evidence suggests one such mechanism for this preferential incorporation of foreign DNA into CRISPR regions during naive CRISPR adaptation is the involvement of the RecBCD complex.…”

Section: Self-targeting With 100% Complementarity Can Drive Evolutionmentioning

confidence: 99%

Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems

Heussler

O’Toole

2016

J Bacteriol

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Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems in bacteria and archaea target foreign elements, such as bacteriophages and conjugative plasmids, through the incorporation of short sequences (termed spacers) from the foreign element into the CRISPR array, thereby allowing sequence-specific targeting of the invader. Thus, CRISPR-Cas systems are typically considered a microbial adaptive immune system. While many of these incorporated spacers match targets on bacteriophages and plasmids, a noticeable number are derived from chromosomal DNA. While usually lethal to the self-targeting bacteria, in certain circumstances, these self-targeting spacers can have profound effects in regard to microbial biology, including functions beyond adaptive immunity. In this minireview, we discuss recent studies that focus on the functions and consequences of CRISPR-Cas self-targeting, including reshaping of the host population, group behavior modification, and the potential applications of CRISPR-Cas self-targeting as a tool in microbial biotechnology. Understanding the effects of CRISPRCas self-targeting is vital to fully understanding the spectrum of function of these systems.

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CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants ofEscherichia coli

Cited by 125 publications

References 58 publications

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

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

Cas9 function and host genome sampling in Type II-A CRISPR–Cas adaptation

Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems

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