Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas Systems

Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR-associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR–Cas systems, contain a single protein, Cas13 (formerly C2c2) that when assembled with a CRISPR RNA (crRNA) forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR–Cas systems can be divided into four subtypes (A–D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference. One RNase is responsible for pre-crRNA processing to form mature Type VI interference complexes, while the other RNase activity provided by the two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, is required for degradation of target-RNA during viral interference. In this review, I will compare and contrast what is known about the molecular architecture and behavior of Type VI (A–D) CRISPR–Cas13 interference complexes, how this allows them to carry out their RNA-targeting function, how Type VI accessory proteins are able to modulate Cas13 activity, and how together all of these features have led to the rapid development of a range of RNA-targeting applications. Throughout I will also discuss some of the outstanding questions regarding Cas13's molecular behavior, and its role in bacterial adaptive immunity and RNA-targeting applications.     

The influence of eukaryotic chromatin state on CRISPR–Cas9 editing efficiencies

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How has horizontal gene transfer shaped the evolution of insect genomes?

As the most diverse group of animals on Earth, insects are key organisms in ecosystems. Horizontal gene transfer (HGT) refers to the transfer of genetic material between species by non-reproductive means. HGT is a major evolutionary force in prokaryotic genome evolution, but its importance in different eukaryotic groups, such as insects, has only recently begun to be understood. Genomic data from hundreds of insect species have enabled the detection of large numbers of HGT events and the elucidation of the functions of some of these foreign genes. Although quantification of the extent of HGT in insects broadens our understanding of its role in insect evolution, the scope of its influence and underlying mechanism(s) of its occurrence remain open questions for the field.     

Underrepresentation of short palindromes in Selenomonas ruminantium DNA: evidence for horizontal gene transfer of restriction and modification systems?

Molecular analysis of isolates of the rumen bacterium Selenomonas ruminantium revealed a high variety and frequency of site-specific (restriction) endonucleases. While all known S. ruminantium restriction and modification systems recognize hexanucleotide sequences only, consistently low counts of both 6-bp and 4-bp palindromes were found in DNA sequences of S. ruminantium. Statistical analysis indicated that there is some correlation between the degree of underrepresentation of tetranucleotide words and the number of known restriction endonucleases for a given sequence. Control analysis showed the same correlation in lambda DNA but not in human adenovirus DNA. Based on the data presented, it could be proposed that there is a much higher historical occurrence of restriction and modification systems in S. ruminantium and (or) frequent horizontal gene transfer of restriction and modification gene complexes.     

Acquisition of freeze protection in a sea-ice crustacean through horizontal gene transfer?

Sea ice is permeated by small brine channels, which are characterised by sub-zero temperatures and varying salinities. Despite sometimes extreme conditions a diverse fauna and flora thrives within the brine channels. The dominant calanoid copepods of Antarctic sea ice are Stephos longipes and Paralabidocera antarctica. Here, I report for the first time thermal hysteresis (TH) in the haemolymph of a crustacean, S. longipes, whereas P. antarctica has no such activity. TH, the non-colligative prevention of ice growth, seems to enable S. longipes to exploit all available microhabitats within sea ice, especially the surface layer, in which strong temperature fluctuations can occur. In contrast, P. antarctica only thrives within the lowermost centimetres of sea ice, where temperature fluctuations are moderate. S. longipes possesses two isoforms of a protein with TH activity. A high homology to a group of (putative) antifreeze proteins from diatoms, bacteria and a snow mold and, in contrast, no homologs in any metazoan lineage suggest that this protein was obtained through horizontal gene transfer (HGT). Further analysis of available sequence data from sea-ice organisms indicates that these antifreeze proteins were probably transferred horizontally several times. Temperature and salinity fluctuations within the brine channel system are proposed to provide “natural transformation” conditions enabling HGT and thus making this habitat a potential “hot spot” for HGT.     

DNA targeting by the type I-G and type I-A CRISPR–Cas systems of Pyrococcus furiosus

CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated.     

Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.     

No inhibition of interferon γ release in human lymphocytes by Ciamexone

Summary In previous experiments Ciamexone, derivative of 2-cyan-aziridine, was able to influence T-cell-mediated regulatory mechanisms but seemed to have no or only little effect on T cell effector mechanisms. On the basis of these observations Ciamexone seems to be a highly selective immunosuppressive agent. In order to evaluate further possible mechanisms of Ciamexone it was the aim of our investigation to study its influence on interferon (IFN) production in phytohaemogglutinin (PHA)-stimulated T lymphocytes of 15 tumour patients and 12 healthy reference subjects. The IFN concentration of the cell supernatant was measured using an enzyme-linked immunosorbent assay. When the cells were stimulated with PHA at 7.5 µg/ml the IFN concentration rose to significantly different values in the reference group (1.0 ng/ml) as compared to the tumour patients (0.4 ng/ml) (P <0.05). An addition of Ciamexone (at any of the concentrations administered) to PHA stimulation of peripheral blood mononuclear cells (PBMC) showed no influence on the IFN release in either test group. The influence of hydrocortisone on the stimulation of PBMC with PHA resulted in a dose-dependent suppression of IFN production in both test groups, again with significant differerences between them. The IFN concentration was 0.95 ng/ml in the reference group and 0.2 ng/ml in the tumour patients when 0.01 µg/ml hydrocortisone was added (P <0.05). At 10 µg/ml hydrocortisone suppressed IFN production completely in both groups. Our results corroborate those investigations that showed no influence of the compound on T cell effector mechanisms. The attenuation of humoral immunophenomena, however, suggest a very specific point of action within the immune system by Ciamexone.     

Does the inhibition of microvillus protein phosphorylation by lysine explain the activity of the latter on calcium transfer?

• 1.1. Is the activity of l-lysine on calcium absorption related to the fact that its phosphorylation is competitive with that of the microvilli proteins involved in the mineral transfer?
• 2.2. The microvilli proteins phosphorylation is not cyclic GMP-dependent but is actually inhibited by l-lysine, used in general at a 100 mM concentration.
• 3.3. The electrophoresis is followed by an autoradiograph which reveals the existence of a phosphorylated protein with a molecular weight of 140,000 daltons. Another phosphorylable protein, clearly visible in some preparations but only detectable in others, has a molecular weight close to 70,000 daltons.
• 4.4. The inhibition by lysine of the microvilli proteins phosphorylation is not specific to a given protein, but is also observed for phosphorylable cytosolic proteins.
• 5.5. A scheme for calcium transfer is proposed. It involves a protein whose phosphorylation should reduce the membrane permeability to calcium.
• 6.6. The following three attributes of the phosphorylable membrane protein—its molecular weight; the fact that another protein (probably its monomer) is also phosphorylable; its well known capacity for phosphorylation—suggest that this protein might actually be alkaline phosphatase whose correlations in calcium metabolism are well known.

     

Knockouts of a late flowering gene via CRISPR–Cas9 confer early maturity in rice at multiple field locations

Plant Molecular Biology - OsGhd7 gene was discovered by screening our rice activation tagging population. CRISPR–Cas9 created knockouts of OsGhd7 conferred early flowering and early maturity...     

Foreign DNA acquisition by the I-F?CRISPR–Cas system requires all components of the interference machinery

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems.     

Analysis of CRISPR–Cas system and antimicrobial resistance in Staphylococcus coagulans isolates

CRISPR–Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR–Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR–Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR–Cas system. Three isolates from Antarctica seals neither contain CRISPR–Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR–Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR–Cas system, and this Type IIIA CRISPR–Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR–Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR–Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.     

No evidence of cellular alterations by MilliTesla-level static and 50 Hz magnetic fields on S. cerevisiae

In an attempt to determine whether magnetic field (MF) exposures might induce cellular alterations, S. cerevisiae yeast cells were exposed to static or sinusoidal 50?Hz homogeneous MF (0.35?mT, 1.4?mT, and 2.45?mT) for 1?h and 72?h. Unsynchronized cells grown exponentially while exposed to MF, containing cells in all stages of the mitotic cell cycle. MF was generated by a pair of Helmholtz coils (40?cm in diameter, coaxial, separated by 20?cm). Survival, cell cycle distribution, colony forming ability, and mutation frequency were assayed. No differences in the above-mentioned parameters were observed in MF exposed samples in relation to unexposed controls, suggesting that homogeneous MF at these intensities do not produce appreciable cellular alterations in this organism under typical in vitro growth conditions.     

Development of an intein-mediated split–Cas9 system for gene therapy

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.     

In silico evidence for the horizontal transfer of gsiB, a σ(B)-regulated gene in gram-positive bacteria, to lactic acid bacteria

gsiB, coding for glucose starvation-inducible protein B, is a characteristic member of the σ(Β) stress regulon of Bacillus subtilis and several other Gram-positive bacteria. Here we provide in silico evidence for the horizontal transfer of gsiB in lactic acid bacteria that are devoid of the σ(Β) factor.     

Detection and characterization of spacer integration intermediates in type I-E CRISPR–Cas system

The adaptation against foreign nucleic acids by the CRISPR–Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5′-ends of the repeat strands with the 3′-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.