CRISPR and Salty: CRISPR-Cas Systems in Haloarchaea

Maier, Lisa‐Katharina; Alkhnbashi, Omer S.; Backofen, Rolf; Marchfelder, Anita

doi:10.1007/978-3-319-65795-0_11

Cited by 5 publications

(6 citation statements)

References 88 publications

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“…Although H. volcanii and H. hispanica are somewhat related haloarchaea their I-B systems are highly divergent, e.g. their PAM preferences differ ( 54 ) and the Cas1 proteins share only 56% identity. It is therefore unsurprising that we show here that in contrast to H. hispanica naïve adaptation is active in H. volcanii.…”

Section: Discussionmentioning

confidence: 99%

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system

Stachler

Wörtz

Alkhnbashi

et al. 2020

Journal of Biological Chemistry

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Haloferax volcanii is to our knowledge the only prokaryote known to tolerate CRISPR-Cas mediated damage to its genome in the wild type background; the resulting cleavage of the genome is repaired by homologous recombination restoring the wild type version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalysed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centred around the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in wild type Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2 and Cas4. Taken together these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.

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Section: Discussionmentioning

confidence: 99%

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system

Stachler

Wörtz

Alkhnbashi

et al. 2020

Journal of Biological Chemistry

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“…Our analysis suggested the presence of several Cas sequences in both wsp1 and wsp4 genomes although no Cas-related genes were identified in the wsp3 genome. In haloarchaea CRISPR-Cas system type I, subtype I-B, is dominantly present ( Maier et al, 2017 ). A characteristic feature of type I systems is the presence of nuclease Cas3 protein, and subtype I-B is further characterized by the presence of Cas8b ( Makarova and Koonin, 2015 ).…”

Section: Resultsmentioning

confidence: 99%

Biochemical and Taxonomic Characterization of Novel Haloarchaeal Strains and Purification of the Recombinant Halotolerant α-Amylase Discovered in the Isolate

et al. 2020

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Haloarchaea are salt-loving archaea and potential source of industrially relevant halotolerant enzymes. In the present study, three reddish-pink, extremely halophilic archaeal strains, namely wsp1 (wsp-water sample Pondicherry), wsp3, and wsp4, were isolated from the Indian Solar saltern. The phylogenetic analysis based on 16S rRNA gene sequences suggests that both wsp3 and wsp4 strains belong to Halogeometricum borinquense while wsp1 is closely related to Haloferax volcanii species. The comparative genomics revealed an open pangenome for both genera investigated here. Whole-genome sequence analysis revealed that these isolates have multiple copies of industrially/biotechnologically important unique genes and enzymes. Among these unique enzymes, for recombinant expression and purification, we selected four putative α-amylases identified in these three isolates. We successfully purified functional halotolerant recombinant Amy2, from wsp1 using pelB signal sequence-based secretion strategy using Escherichia coli as an expression host. This method may prove useful to produce functional haloarchaeal secretory recombinant proteins suitable for commercial or research applications. Biochemical analysis of Amy2 suggests the halotolerant nature of the enzyme having maximum enzymatic activity observed at 1 M NaCl. We also report the isolation and characterization of carotenoids purified from these isolates. This study highlights the presence of several industrially important enzymes in the haloarchaeal strains which may potentially have improved features like stability and salt tolerance suitable for industrial applications.

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“…Owing to strong conservation of haloarchaeal CRISPR repeat sequences, it is highly likely that a CRISPR locus acquired via horizontal gene transfer can be utilized by the host CRISPR-Cas system. Despite the danger of acquiring suicidal spacers, the overwhelming majority of haloarchaeal genomes retain a functional CRISPR-Cas system [16]. The implications of targeting an actively transcribed, yet non-essential, chromosomal gene on cellular fitness have recently been studied in H. volcanii [24].…”

Section: Self-targeting Is Tolerated In H Volcaniimentioning

confidence: 99%

“…With a few exceptions, archaeal CRISPR-Cas systems are class 1 systems characterized by multi-protein effector complexes [14, 15]. Class 1 comprises a plethora of types and subtypes whose biochemical and mechanistic diversity can be substantial even within species and subtypes, e.g., as seen for the halobacterial type I-B systems [13, 16, 17]. These individual peculiarities make it difficult to draw a general conclusion from studying subtypes in only one species and illustrate how important a broad spectrum of studied organisms is for discerning common features.…”

Section: Introducing the Key Players: Cas Proteins And Crispr Rnamentioning

confidence: 99%

“…The diversity of CRISPR-Cas systems within archaea is quite high, and often more than one CRISPR-Cas system is present in a genome [15]. H. volcanii is a euryarchaeon, and within this clade, almost all varieties of the type I and type III subtypes can be found [15, 16]. However, the halophilic archaea of the class Halobacteriales are quite homogenous in their CRISPR content since they contain only type I-B and I-D systems [16].…”

Section: Introducing the Key Players: Cas Proteins And Crispr Rnamentioning

confidence: 99%

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The nuts and bolts of the Haloferax CRISPR-Cas system I-B

et al. 2018

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Invading genetic elements pose a constant threat to prokaryotic survival, requiring an effective defence. Eleven years ago, the arsenal of known defence mechanisms was expanded by the discovery of the CRISPR-Cas system. Although CRISPR-Cas is present in the majority of archaea, research often focuses on bacterial models. Here, we provide a perspective based on insights gained studying CRISPR-Cas system I-B of the archaeon Haloferax volcanii. The system relies on more than 50 different crRNAs, whose stability and maintenance critically depend on the proteins Cas5 and Cas7, which bind the crRNA and form the Cascade complex. The interference machinery requires a seed sequence and can interact with multiple PAM sequences. H. volcanii stands out as the first example of an organism that can tolerate autoimmunity via the CRISPR-Cas system while maintaining a constitutively active system. In addition, the H. volcanii system was successfully developed into a tool for gene regulation.

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CRISPR and Salty: CRISPR-Cas Systems in Haloarchaea

Cited by 5 publications

References 88 publications

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system

Biochemical and Taxonomic Characterization of Novel Haloarchaeal Strains and Purification of the Recombinant Halotolerant α-Amylase Discovered in the Isolate

The nuts and bolts of the Haloferax CRISPR-Cas system I-B

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