Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic <i>Sulfolobales</i> and the evasion of lethal gene silencing

Zink, Isabelle Anna; Fouqueau, Thomas; Risa, Gabriel Tarrason; Werner, Finn; Baum, Buzz; Bläsi, Udo; Schleper, Christa

doi:10.1080/15476286.2020.1813411

Cited by 12 publications

(8 citation statements)

References 79 publications

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“…No c-tetra-AMP could be detected in any of the samples prepared for this study. As S. acidocaldarius encodes a functional type III CRISPR system that contains a Cas10 subunit ( Zink et al, 2021 ) detection of c-tetra-AMP in extracts from this species could have been expected. However, as the cells in this study were not challenged with any invading virus it is plausible that the observed lack of c-tetra-AMP originates from the type III CRISPR system of S. acidocaldarius being inactive at the tested conditions.…”

Section: Resultsmentioning

confidence: 99%

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

et al. 2021

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Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3′,5′-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3′,5′-cAMP, cyclic guanosine monophosphate (3′,5′-cGMP), 5′-phosphoadenylyl-3′,5′-adenosine (5′-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2′,3′-cyclic isomers of all four RNA building blocks (2′,3′-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3′,5′-cCMP), cyclic uridine monophosphate (3′,5′-cUMP) and cyclic diadenosine monophosphate (3′,5′-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3′,5′-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

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Section: Resultsmentioning

confidence: 99%

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

et al. 2021

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“…Higher silencing levels conferred by stronger spacers (or an increased numbers of otherwise stable spacers) were not tolerated, leading to precise excision of those spacers from the miniCRISPR array. Thus, this suggests the presence of a probably CRISPR-linked mechanism to eradicate deleterious spacers [198,237].…”

Section: Crispr Application In Archaeal Modelsmentioning

confidence: 92%

“…A native CRISPR type III system can be repurposed for posttranscriptional silencing of host genes in S. solfataricus [119,249], S. islandicus [118,250] and S. acidocaldarius [237]. For type III-mediated silencing, crRNAs are heterologously expressed from a miniCR vector, incorporated into a native CRISPR type III endonuclease and guided to complementary loci on the mRNA of a desired gene which is subsequently cleaved [118,119].…”

Section: Crispr Application In Archaeal Modelsmentioning

confidence: 99%

“…In theory, this technology can readily be applied in any genetically accessible organism carrying a type III system, as no genetic manipulation of the complex is required beforehand. Besides some non-essential genes that could be silenced to almost 100% [118,249,250], we have recently applied the type III-mediated knockdown on essential genes of different functional categories, including cell division, transcription, cell wall biogenesis and translation [237,251,252]. We found that dependent on the gene, a maximum silencing efficiency of 40-75% was achieved and could not be exceeded [237].…”

Section: Crispr Application In Archaeal Modelsmentioning

confidence: 99%

“…Besides some non-essential genes that could be silenced to almost 100% [118,249,250], we have recently applied the type III-mediated knockdown on essential genes of different functional categories, including cell division, transcription, cell wall biogenesis and translation [237,251,252]. We found that dependent on the gene, a maximum silencing efficiency of 40-75% was achieved and could not be exceeded [237]. Within this range, the silencing effect was stable and specific phenotypes could be analyzed in vivo, allowing functional characterization of the respective gene [251].…”

Section: Crispr Application In Archaeal Modelsmentioning

confidence: 99%

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Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

2020

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Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

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Reprogramming CRISPR-Mediated RNA Interference for Silencing of Essential Genes in Sulfolobales

Wimmer

Zink

Schleper

2022

Archaea

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Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing

Cited by 12 publications

References 79 publications

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Reprogramming CRISPR-Mediated RNA Interference for Silencing of Essential Genes in Sulfolobales

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