Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering.

Microbial systems underpin many biotechnologies, including CRISPR, but the exponential growth of sequence databases makes it difficult to find previously unidentified systems. In this work, we develop the fast locality-sensitive hashing-based clustering (FLSHclust) algorithm, which performs deep clustering on massive datasets in linearithmic time. We incorporated FLSHclust into a CRISPR discovery pipeline and identified 188 previously unreported CRISPR-linked gene modules, revealing many additional biochemical functions coupled to adaptive immunity. We experimentally characterized three HNH nuclease-containing CRISPR systems, including the first type IV system with a specified interference mechanism, and engineered them for genome editing. We also identified and characterized a candidate type VII system, which we show acts on RNA. This work opens new avenues for harnessing CRISPR and for the broader exploration of the vast functional diversity of microbial proteins.

The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity.

TnpB is a member of the Obligate Mobile Element Guided Activity (OMEGA) RNA-guided nuclease family, is harbored in transposons, and likely functions to maintain the transposon in genomes. Previously, it was shown that TnpB cleaves double- and single-stranded DNA substrates in an RNA-guided manner, but the biogenesis of the TnpB ribonucleoprotein (RNP) complex is unknown. Using in vitro purified apo TnpB, we demonstrate the ability of TnpB to generate guide omegaRNA (ωRNA) from its own mRNA through 5' processing. We also uncover a potential cis-regulatory mechanism whereby a region of the TnpB mRNA inhibits DNA cleavage by the RNP complex. We further expand the characterization of TnpB by examining ωRNA processing and RNA-guided nuclease activity in 59 orthologs spanning the natural diversity of the TnpB family. This work reveals a new functionality, ωRNA biogenesis, of TnpB, and characterizes additional members of this biotechnologically useful family of programmable enzymes.

Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.

Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB-ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA-target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB-ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes.

RNA-activated protein cleavage with a CRISPR-associated endopeptidase.

In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA.

RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies1,2. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development3. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain4. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.

The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases.

IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element­guided activity), with strong potential for developing as biotechnologies.

Structural Analysis of Different LINC Complexes Reveals Distinct Binding Modes.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.

The AAA + ATPase TorsinA polymerizes into hollow helical tubes with 8.5 subunits per turn.

TorsinA is an ER-resident AAA + ATPase, whose deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is an unusual AAA + ATPase that needs an external activator. Also, it likely does not thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of its still enigmatic function. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 Å structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner channel of ~ 4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA + ATPases. We discuss the implications of these observations for TorsinA function.

Structures of TorsinA and its disease-mutant complexed with an activator reveal the molecular basis for primary dystonia.

The most common cause of early onset primary dystonia, a neuromuscular disease, is a glutamate deletion (ΔE) at position 302/303 of TorsinA, a AAA+ ATPase that resides in the endoplasmic reticulum. While the function of TorsinA remains elusive, the ΔE mutation is known to diminish binding of two TorsinA ATPase activators: lamina-associated protein 1 (LAP1) and its paralog, luminal domain like LAP1 (LULL1). Using a nanobody as a crystallization chaperone, we obtained a 1.4 Å crystal structure of human TorsinA in complex with LULL1. This nanobody likewise stabilized the weakened TorsinAΔE-LULL1 interaction, which enabled us to solve its structure at 1.4 Å also. A comparison of these structures shows, in atomic detail, the subtle differences in activator interactions that separate the healthy from the diseased state. This information may provide a structural platform for drug development, as a small molecule that rescues TorsinAΔE could serve as a cure for primary dystonia.

Purification and Structural Analysis of SUN and KASH Domain Proteins.

Molecular tethers span the nuclear envelope to mechanically connect the cytoskeleton and nucleoskeleton. These bridge-like tethers, termed linkers of nucleoskeleton and cytoskeleton (LINC) complexes, consist of SUN proteins at the inner nuclear membrane and KASH proteins at the outer nuclear membrane. LINC complexes are central to a variety of cell activities including nuclear positioning and mechanotransduction, and LINC-related abnormalities are associated with a spectrum of tissue-specific diseases, termed laminopathies or envelopathies. Protocols used to study the biochemical and structural characteristics of core elements of SUN-KASH complexes are described here to facilitate further studies in this new field of cell biology.

The association of obesity and obesity-related conditions with carotid extra-medial thickness in children and adolescents.

BACKGROUND: Carotid extra-medial thickness (EMT) and carotid intima-media thickness (cIMT) provide information concerning vascular changes. OBJECTIVES: In this study, we aimed to evaluate the association between carotid EMT and obesity and its metabolic complications in children. METHODS: The study included 38 obese subjects and 30 age-matched and sex-matched healthy controls aged between 7 and 17 years. For all subjects, complete blood count, fasting blood glucose, serum insulin, aspartate aminotransferase, alanine aminotransferase, HDL cholesterol, total cholesterol and triglyceride levels were measured. The carotid EMT and cIMT were measured by an expert radiologist in all patients. RESULTS: Body mass index (BMI) (28.8 ± 3 vs. 18.1 ± 2.2, p < 0.001), total cholesterol (167.9 ± 34.8 mg dL-1 vs. 150.5 ± 28.1 mg dL-1 , p = 0.029), homeostatic model assessment of insulin resistance (HOMA-IR) (4.3 vs. 1.7, p < 0.001), cIMT (0.51 ± 0.08 mm vs. 0.45 ± 0.06 mm, p < 0.001) and carotid EMT (0.74 ± 0.11 mm vs. 0.64 ± 0.1 mm, p < 0.001) were significantly higher in obese subjects than in controls, while HDL cholesterol (41.6 ± 6.5 mg dL-1 vs. 49.5 ± 7.5 mg dL-1 , p < 0.001) was lower in obesity group. Among the obese subjects, the HOMA-IR values (4.7 vs. 3.6, p = 0.027), cIMT (0.54 ± 0.07 mm vs. 0.49 ± 0.07 mm, p = 0.039) and carotid EMT (0.79 ± 0.1 mm vs. 0.7 ± 0.1 mm, p = 0.013) were significantly higher in post-pubertal children compared with prepubertal children. BMI, cut-off values of HOMA-IR and cIMT were significantly associated with increased carotid EMT (p < 0.001, p = 0.023 and p < 0.001, respectively). The only independent risk factor affecting carotid EMT was BMI (p < 0.001). CONCLUSION: We have found that carotid EMT is associated with cIMT, obesity and insulin resistance and the assessment of carotid EMT may provide additional information concerning early vascular disease.

Neutrophil-to-lymphocyte ratio and red blood cell distribution width is a practical predictor for differentiation of febrile seizure types.

OBJECTIVE: Febrile seizures (FS) are the most common neurological emergency in childhood. They are divided into two groups accordingly clinical features, simple febrile seizure and complex febrile seizure. Until now laboratory tests have not been used as a parameter of classification of them. The objective of this study is to estimate the usefulness of the hematogical parameters especially neutrophil-to-lymphocyte ratio (NLR) and red blood cell distribution width (RDW) in the differentiation of febrile seizure types. PATIENTS AND METHODS: A retrospective review was conducted on patients from 6 months to 6 years old presenting with first febrile seizure admitted to a tertiary care hospital. Epidemiological and laboratory variables of the patients were collected. RESULTS: The mean NLR in the simple FS and complex FS groups was 2.18±1.9 and 3.8±4.2 respectively, and the difference was significant (p=0.024). The mean serum red blood cell distribution width in the simple FS and complex FS groups was 16.1±1.1 and 16.6±0.8 respectively, and the difference was significant (p=0.019). NLR and RDW values in complex FS patients were statistically higher than simple febrile patients. ROC analysis showed that if the chosen cut-off point for NLR is 1.98 the sensitivity and specificity are 66.7% and 60.3% respectively. These were statistically significant (p=0.040 AUC 0.623, CI 0.503-0.743). If the chosen cut-off point for RDW is 16.350, the sensitivity and specificity are 59.0% and 58.6%, respectively. These were statistically significant (p=0.037 AUC 0.626, CI 0.515-0.736) too. CONCLUSIONS: NLR and RDW were simple, effective and practical predictors for differentiation of FS types. They will have potential values in public health practice for management of FS patients.

The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ?closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.

How lamina-associated polypeptide 1 (LAP1) activates Torsin.

Lamina-associated polypeptide 1 (LAP1) resides at the nuclear envelope and interacts with Torsins, poorly understood endoplasmic reticulum (ER)-localized AAA+ ATPases, through a conserved, perinuclear domain. We determined the crystal structure of the perinuclear domain of human LAP1. LAP1 possesses an atypical AAA+ fold. While LAP1 lacks canonical nucleotide binding motifs, its strictly conserved arginine 563 is positioned exactly where the arginine finger of canonical AAA+ ATPases is found. Based on modeling and electron microscopic analysis, we propose that LAP1 targets Torsin to the nuclear envelope by forming an alternating, heterohexameric (LAP1-Torsin)3 ring, in which LAP1 acts as the Torsin activator. The experimental data show that mutation of arginine 563 in LAP1 reduces its ability to stimulate TorsinA ATPase hydrolysis. This knowledge may help scientists understand the etiology of DYT1 primary dystonia, a movement disorder caused by a single glutamate deletion in TorsinA.

Hepatic artery resistance in children with obesity and fatty liver.

OBJECTIVE: The present study assessed whether there is a correlation between hepatic artery resistive index (HARI) and increase of body mass index and hepatosteatosis grading in children with non-alcoholic fatty liver disease (NAFLD) reflecting hemodynamic effects of hepatosteatosis. METHODS: Thirty three healthy children [body mass index(BMI): mean+/- standart deviation(SD), min-max: 20.1+/-1.14(18.5-23.7), 33 overweight [BMI:25.1+/-2.2 (18.5-23.7)] and 66 obese [BMI:31.1+/-2(25.6-40)] adolescents were enrolled into the study. To search the relation of HARI with fatty liver degree, study subjects subdivided into groups according to their degree of fatty liver at ultrasonography(US). RESULTS: Increase of HARI was correlated with increase in BMI (p<0.0001, r=0.533). Increase of HARI was well correlated with increase in degree of fatty liver (p<0.0001, r=0.630). CONCLUSION: The present study results suggest that there are positive correlations of HARI with BMI and hepatosteatosis grade in obese children with NAFLD. HARI may be a candidate parameter to determine early alarming hemodynamic changes in hepatic tissue of obese children with fatty liver before development of severe stages NAFLD.

Leukocyte phagocytosis during cardiopulmonary bypass in adolescents.

This study was performed to determine the absolute PMNL count as well as phagocytic functions in adolescents who had undergone CPB; a radioactive method was used for the first time in this field. Although CPB causes a decrease in the absolute PMNL count when the subject is off bypass, this value exceeded the baseline level within three days. A day prior to surgery PMNL phagocytosis was found to be normal and was unaffected within the first minutes on bypass. Whereas, PMNL phagocytosis decreased significantly off bypass. However, the decrease was transient and returned to normal within three days. Another interesting finding obtained as a result of this study was that in the cases in whom the clinical infection picture was seen, the patient had a longer duration on bypass and a higher inhibition of leukocyte phagocytosis. The correlation between the duration on bypass and inhibition of leukocyte phagocytosis could be shown as a trend. Thus it may be concluded that the longer the on bypass period the higher the inhibition of leukocytic phagocytosis and the higher the infection rate in the postoperative period.