Genetic technologies for extremely thermophilic microorganisms of Sulfolobus, the only genetically tractable genus of crenarchaea

Peng, Nan; Han, Wenyuan; Li, Yingjun; Liang, Yunxiang; She, Qunxin

doi:10.1007/s11427-016-0355-8

Cited by 59 publications

(44 citation statements)

References 150 publications

(203 reference statements)

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“…Here, we present the development of a thermostable protein tag suitable for protein visualization in the hyperthermophilic crenarcheaon S . islandicus , one of the most useful archaeal models for genetic studies [ 28 ]. To this aim, we constructed, by a CRISPR-based genome-editing method [ 29 ], a S .…”

Section: Introductionmentioning

confidence: 99%

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

et al. 2017

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Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

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Section: Introductionmentioning

confidence: 99%

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

et al. 2017

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“…The altered spacers were cloned into the Sulfolobus expression vector pSeSD (35) to generate the plasmid-borne mini-CRISPRs under the control of the arabinose-inducible promoter (Fig. 6C) (32, 36). These challenging plasmids were then transformed into S. islandicus E233S cells and transformation efficiencies were estimated.…”

Section: Resultsmentioning

confidence: 99%

“…S. islandicus REY15A has proven to be a successful model organism for studying crenarchaeal CRISPR–Cas systems. It carries one subtype I-A and two functionally different subtype III-B interference modules that share an adaptation module encoded adjacent to the subtype I-A interference module (31) and, importantly, a comprehensive array of genetic tools has been developed for this strain (26, 32). Recently, we demonstrated that Csa3a, the activator protein of the subtype I-A CRISPR-Cas immune response in S. islandicus , couples transcriptional activation of spacer acquisition and DNA repair genes and, moreover, it enhances CRISPR-Cas interference of invading genetic elements by activating transcription of CRISPR arrays (9, 19).…”

Section: Introductionmentioning

confidence: 99%

Cas4 nucleases can effect specific integration of CRISPR spacers

Zhang

Pan

Liu

et al. 2018

Preprint

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13CRISPR-Cas systems incorporate short DNA fragments from invasive genetic elements into 14 host CRISPR arrays in order to generate host immunity.Recently, we demonstrated that the 15 Csa3a regulator protein triggers CCN PAM-dependent CRISPR spacer acquisition in the 16 subtype I-A CRISPR-Cas system of Sulfolobus islandicus. However, the mechanisms 17 underlying specific protospacer selection and spacer insertion remained unclear. Here, we 18 demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (a) in 19 recognizing the 5΄ PAM and 3΄ nucleotide motif of protospacers and (b) in determining both 20 the spacer length and its orientation. Furthermore, we identify uncovered amino acid residues 21 of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 22 proteins, and also of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced 23 adaptation efficiency and the formers yielded a high incidence of PAM-dependent atypical 24 spacer integration, or of PAM-independent spacer integration. We further demonstrated that, 25 in the plasmid challenging experiments, overexpressed Cas4-mediated defective spacer 26 acquisition, in turn, potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas 27 interference. In summary, these results define the specific involvement of diverse Cas4 28 proteins in in vivo CRISPR spacer acquisition. Furthermore, we provide support for an anti-29 CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas 30 interference activity by hindering spacer acquisition. 31 Importance 33The Cas4 family endonuclease is an essential component of the adaptation module in many 34 variants of CRISPR-Cas adaptive immunity systems. The Crenarchaeota Sulfolobus 35 islandicus REY15A encodes two cas4 genes (cas4 and csa1) linked to the CRISPR arrays. 36Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this 37 organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of 38 the protospacers and define the spacer length and orientation in the acquisition process. 39Conserved amino acid residues, in addition to the recently reported, were identified to be 40 important for above functions. More importantly, overexpression of the Sulfolobus viral Cas4 41 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded 42 Cas4 proteins that inhibit spacer acquisition. 43 44 bacteria that target invasive viruses and plasmids (1, 2). These diverse systems have been 48 classified into two major classes and at least 6 basic types (Types I to VI) that are further 49 divided into multiple subtypes (3). Spacer acquisition into CRISPR arrays constitutes the first 50 stage of the immune reaction (4); however, the molecular mechanisms involved in this 51 process are still only partially understood. Short DNA fragments of similar length are excised 52 from the invasive genetic element and integrated into the CRISPR loci facilitated by the 53...

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“…6C) spacers were cloned into the Sulfolobus expression vector pSeSD (35) to generate plasmid-borne mini-CRISPRs under the control of the arabinose-inducible promoter ( Fig. 6C) (32,36). These challenging plasmids were then transformed into S. islandicus E233S cells, and transformation efficiencies were estimated.…”

Section: Crisprlinked Cas4mentioning

confidence: 99%

Cas4 Nucleases Can Effect Specific Integration of CRISPR Spacers

et al. 2019

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Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity. Recently, we demonstrated that the Csa3a regulator protein triggers CCN protospacer-adjacent motif (PAM)-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus. However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (i) in recognizing the 5= PAM and 3= nucleotide motif of protospacers and (ii) in determining both the spacer length and its orientation. Furthermore, we identify amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also that of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency, and the former proteins yielded a high incidence of PAM-dependent atypical spacer integration or of PAM-independent spacer integration. We further demonstrated that in plasmid challenge experiments, overexpressed Cas4mediated defective spacer acquisition in turn potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas interference. In summary, these results define the specific involvement of diverse Cas4 proteins in in vivo CRISPR spacer acquisition. Furthermore, we provide support for an anti-CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas interference activity by hindering spacer acquisition. IMPORTANCE The Cas4 family endonuclease is an essential component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. The Crenarchaeota Sulfolobus islandicus REY15A carries two cas4 genes (cas4 and csa1) linked to the CRISPR arrays. Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of the protospacers and define the spacer length and orientation in the acquisition process. Conserved amino acid residues, in addition to those recently reported, were identified to be important for these functions. More importantly, overexpression of the Sulfolobus viral Cas4 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded Cas4 proteins that inhibit spacer acquisition.

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Genetic technologies for extremely thermophilic microorganisms of Sulfolobus, the only genetically tractable genus of crenarchaea

Cited by 59 publications

References 150 publications

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

Cas4 nucleases can effect specific integration of CRISPR spacers

Cas4 Nucleases Can Effect Specific Integration of CRISPR Spacers

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