Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis

Shmakov, Sergey; Makarova, Kira S.; Wolf, Yuri I.; Severinov, Konstantin; Koonin, Eugene V.

doi:10.1073/pnas.1803440115

Cited by 145 publications

(170 citation statements)

References 109 publications

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“…Consistent with earlier results 1,8 , only a small fraction of spacers yielded significant matches: ~12% and ~7%, for type IV and non-type IV, respectively ( Fig. 3a, Supplementary Table 4 and Supplementary Data 3 and 4), which reflects the current undersampling of the microbiome 22 .…”

Section: Type IV Spacer Contents Exhibit a Strong Bias Towards Plasmisupporting

confidence: 90%

“…Moreover, subtype IV-A loci encode a DinG family helicase (Csf4), a type IV-specific Cas6-like protein (Csf5), and they typically colocate with a CRISPR array. In contrast, subtype IV-B loci lack dinG, csf5 and an associated CRISPR array but they encode a putative "small subunit" (Cas11) and they often neighbour a cysH gene 7,8 . A recent structural and biochemical analysis of a subtype IV-A CRISPR-Cas system demonstrated the essential role of the Cas6-like enzyme in both the maturation of crRNAs and in the subsequent formation of a Cascade-like crRNA-guided effector complex, composed of Csf1, Csf3, Csf5 and multiple copies of Csf2 9 .…”

Section: Introductionmentioning

confidence: 99%

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Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

Pinilla‐Redondo

Mayo-Muñoz

Russel

et al. 2019

Preprint

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CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites by leveraging small non-coding RNAs for nuclease-dependent degradation of their nucleic acid targets. In contrast to all other types of CRISPR-Cas systems, the mechanisms and biological roles of type IV systems have remained largely overlooked.Here, we describe a previously uncharted diversity of type IV gene cassettes, distributed across diverse prokaryotic genome backgrounds, and propose their classification into subtypes and variants. Congruent with recent findings, type IV modules were primarily found on plasmid-like elements. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a yet unrecognised role in mediating conflicts between plasmids that compete to monopolize their hosts. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing an answer to the enigmatic absence of adaptation modules in these systems. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.

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Section: Type IV Spacer Contents Exhibit a Strong Bias Towards Plasmisupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

Pinilla‐Redondo

Mayo-Muñoz

Russel

et al. 2019

Preprint

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“…For instance, Shmakov et al . systematically identified Cas‐associated genes by determining CRISPR‐Cas systems and (sub)types ; Shah et al . identified some co‐functional accessory genes within the type III CRISPR‐Cas system using a guilt‐by‐association approach and Makarova et al .…”

Section: Discussionmentioning

confidence: 99%

CasLocusAnno: a web‐based server for annotating cas loci and their corresponding (sub)types

Dong

Zeng

et al. 2019

FEBS Letters

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In prokaryotes, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‐associated protein (Cas) systems constitute adaptive immune systems against mobile genetic elements (MGEs). Here, we introduce the Markov cluster algorithm (MCL) to Makarova et al.'s method in order to select a more reasonable profile. Additionally, our new Maximum Continuous Cas Subcluster (MCCS) method helps identification of tightly clustered loci. The comparison with two other commonly used programs shows that the method could identify Cas proteins with higher accuracy and lower Additional Prediction Rate (APR). Moreover, we developed a web‐based server, CasLocusAnno (http://cefg.uestc.cn/CasLocusAnno), capable of annotating Cas proteins, cas loci and their (sub)types less than ~ 28 s following the whole proteome sequence submission. Its standalone version can be downloaded at https://github.com/RiversDong/CasLocusAnno.

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“…emphasizing the importance of cOA signalling for type III CRISPR defence (Jiang et al, 2016, 44 Grüschow et al, 2019, Foster et al, 2019, Deng et al, 2013). A wide variety of alternative CARF-45 domain proteins associated with type III CRISPR loci have been identified but not yet described 46 (Shmakov et al, 2018, Shah et al, 2018, and recent work has characterized the cA 4 -activated 47…”

Section: Introduction 23mentioning

confidence: 99%

“…Ring nucleases thus appear to be an important constituent of virus:host conflict in the expanding 70 arena of cyclic nucleotide signalling. Here, we focus on the Csx3 family of proteins, which is found 71 associated with many type III CRISPR systems (Shah et al, 2018, Shmakov et al, 2018. Csx3 72…”

Section: Introduction 23mentioning

confidence: 99%

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Graham

Grüschow

Gloster

et al. 2020

Preprint

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9Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to 11 signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This 12 can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell 13once a viral infection has been defeated. Enzymes specialised for this task are known as ring 14nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR 15 associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that 16rapidly degrades cyclic tetra-adenylate (cA 4 ). The enzyme has an unusual cooperative reaction 17 mechanism involving an active site that spans the interface between two dimers, sandwiching the 18 cA 4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 19family. 20 Keywords: 21 CRISPR, Csx3, ring nuclease, cyclic tetra-adenylate, CARF, cooperative enzyme 22 103 terminal Csx3 domain fused to a C-terminal kinase/transferase domain of unknown function. B. Phosphor 104 images of native gel electrophoresis visualising cA 4 (20 nM) or RNA oligonucleotide 49-9A (50 nM) binding 105 by A. fulgidus Csx3. Csx3 binds to cA 4 with high affinity (apparent K D ~ 50 nM) and binds the RNA 49-9A 106 with significantly lower affinity (apparent K D ~ 10 µM). Images are representative of three technical 107 replicates. 108 109 Figure 1figure supplement 1. Multiple sequence alignment of Csx3 proteins showing conserved 110 residues. Csx3 proteins are shown from Archaeoglobus fulgidus; Methanosarcinia mazei; candidatus 111 Bathyarchaeota; Thermococcus celericrescens; Methylacidiphilum fumariolicum; Aquifex aeolicus; 112 Dictyoglomus turgidum; Spirulina major; Oscillatoria nigro-viridis. Absolutely conserved residues are shaded. 113 Conserved residues H60 and D69 (Afu numbering) are indicated by asterisks. 114 131 Figure 2. Csx3 is a potent ring nuclease. A. TLC analysis of the reaction products of radiolabelled cA 4 132 (200 nM) incubated with A. fulgidus Csx3 (8 µM dimer) in reaction buffer at 50 °C (time points every 5 s from 133 10-60 s then 1.5, 2, 3, 4, 5 and 10 min, n = 6 technical replicates). The cA 4 was rapidly converted into A 2 -P, 134 with a small amount of A 2 >P (linear A 2 with a cyclic 2',3' terminal phosphate). B. Denaturing polyacrylamide 135 gel electrophoresis visualising cleavage of 5'-end radiolabelled RNA 49-9A (50 nM) by Csx3 (8 µM dimer) at 136 50 °C (time points every 5 min from 5-40 min then 50, 60 and 90 min, n = 3 technical replicates). C. Plot 137 comparing single-turnover kinetics of cA 4 (blue) and RNA 49-9A (green) cleavage by AfCsx3 at 50 °C. Data 138 points are the average of three technical replicates and error bars represent the standard deviation of the 139 mean. D. Liquid-chromatography high-resolution mass spectrometry analysis of reaction products when cA 4

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Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis

Cited by 145 publications

References 109 publications

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

CasLocusAnno: a web‐based server for annotating cas loci and their corresponding (sub)types

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

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