Structures and mechanisms of tRNA methylation by METTL1-WDR4.

Specific, regulated modification of RNAs is important for proper gene expression1,2. tRNAs are rich with various chemical modifications that affect their stability and function3,4. 7-Methylguanosine (m7G) at tRNA position 46 is a conserved modification that modulates steady-state tRNA levels to affect cell growth5,6. The METTL1-WDR4 complex generates m7G46 in humans, and dysregulation of METTL1-WDR4 has been linked to brain malformation and multiple cancers7-22. Here we show how METTL1 and WDR4 cooperate to recognize RNA substrates and catalyse methylation. A crystal structure of METTL1-WDR4 and cryo-electron microscopy structures of METTL1-WDR4-tRNA show that the composite protein surface recognizes the tRNA elbow through shape complementarity. The cryo-electron microscopy structures of METTL1-WDR4-tRNA with S-adenosylmethionine or S-adenosylhomocysteine along with METTL1 crystal structures provide additional insights into the catalytic mechanism by revealing the active site in multiple states. The METTL1 N terminus couples cofactor binding with conformational changes in the tRNA, the catalytic loop and the WDR4 C terminus, acting as the switch to activate m7G methylation. Thus, our structural models explain how post-translational modifications of the METTL1 N terminus can regulate methylation. Together, our work elucidates the core and regulatory mechanisms underlying m7G modification by METTL1, providing the framework to understand its contribution to biology and disease.

Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.

The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.

Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.

The bridge helix of Cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage.

Cas12a is an RNA-guided DNA endonuclease of the type V-A CRISPR-Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P ) to test mechanistic conservation. Our results show that for RNA-guided DNA cleavage (cis-activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans-single-stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis- and trans-cleavages and show its conserved role in target discrimination among Cas nucleases.

Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain.

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.

Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance.

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.

The structure of DLP12 endolysin exhibiting alternate loop conformation and comparative analysis with other endolysins.

The lytic enzyme, endolysin, is encoded by bacteriophages (phages) to destroy the peptidoglycan layer of host bacterial cells. The release of phage progenies to start the new infection cycle is dependent on the cell lysis event. Endolysin encoded by DLP12 cryptic prophage is a SAR endolysin which is retained by the bacterium presumably due to the benefit it confers. The structure of DLP12 endolysin (Id: 4ZPU) determined at 2.4 Å resolution is presented here. The DLP12 endolysin structure shows a modular nature and is organized into distinct structural regions. One of the monomers has the loops at the active site in a different conformation. This has led to a suggestion of depicting possibly active and inactive state of DLP12 endolysin. Comparison of DLP12 endolysin structure and sequence with those of related endolysins shows the core three-dimensional fold is similar and the catalytic triad geometry is highly conserved despite the sequence differences. Features essential for T4 lysozyme structure and function such as the distance between catalytic groups, salt bridge and presence of nucleophilic water are conserved in DLP12 endolysin and other endolysins analyzed.

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.

CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems.

Umeclidinium in chronic obstructive pulmonary disease: latest evidence and place in therapy.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity, mortality and health care expenditure throughout the world. COPD guidelines recommend the use of long-acting muscarinic antagonist (LAMA) either alone or in combination with a long-acting ß2 agonist (LABA). For over 10 years, tiotropium was the only LAMA that was used in the management of COPD. Over the past few years, various new drugs have been identified that act on the muscarinic receptors and ß2 receptors. Umeclidinium (Umec) is a new LAMA currently approved for use in patients with COPD either as monotherapy or in combination with vilanterol (Vil). Both Umec alone and in combination with Vil delivered through a multi-dose dry powder Ellipta™ device have shown improvement in lung function, health-related quality of life and exacerbation frequency in patients with COPD. This review provides an overview of the pharmacology, pharmacodynamics and pharmacokinetics of Umec, and evaluates the clinical efficacy and safety studies in patients with COPD.

Conserved DNA motifs in the type II-A CRISPR leader region.

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3' end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3' leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3' leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

Investigational beta-2 adrenergic agonists for the treatment of chronic obstructive pulmonary disease.

INTRODUCTION: Long-acting bronchodilators are pivotal in the therapeutic management of COPD patients with moderate-to-severe airflow obstruction. New ultra-long-acting ß2-agnoists (ultra-LABAs) have been developed, some of which have been licensed for use as monotherapy and/or in combination with other bronchodilators or inhaled corticosteroids, for use in COPD patients with persistent symptoms and worsening airflow limitation. These new agents are faster in onset and have a prolonged duration of action, with a similar safety profile to the traditional twice-daily bronchodilators which may have an impact on patient concordance. Areas covered: A number of these ultra-LABAs are still under development and bi-functional hybrid molecules containing regions functioning as ß2-agonists, and as muscarinic agonists (MABAs) has been developed. This review summarizes these (excluding the licensed ultra-LABAs) with attention on phase II studies data available to-date on their pharmacological profiles, clinical efficacy and safety, and future perspectives. Expert opinion: Despite all the new agents' available, the challenges that persist include any differences in efficacy and safety between the various possible LAMA/LABA combinations, relative advantages of MABAs over fixed-dose LAMA/LABAs, and the impact of these new molecules in terms of long term safety, especially in certain populations in co-morbidities frequently associated with COPD.

Emerging therapeutic strategies in COPD.

Chronic obstructive pulmonary disease (COPD) management is changing with the advent of newer inhaled medications and devices that belong to the current group of therapies [i.e. inhaled corticosteroid (ICS), long-acting ß2 agonists (LABAs) and long-acting muscarinic antagonists (LAMAs)], providing patients as well as physicians with a wider range of options. In this review, we examine Phase II studies currently underway in COPD patients. With improved understanding of the condition, the role for biological and immunomodulatory therapies in COPD patients is also an interesting and important aspect looked upon with great enthusiasm. It is indeed interesting to note that the future COPD treatment options could include novel interventional strategies in addition to innovative inhaled therapies, which have been the backbone of COPD management for the past couple of decades.

Anti-IgE in allergic asthma and rhinitis: an update.

Allergic asthma and rhinitis imposes a huge burden in terms of treatment costs, productivity loss and hospital admissions. IgE plays a significant role in the manifestation of these conditions and the identification of a monoclonal antibody that binds to IgE provides clinicians another therapeutic strategy in the management of these conditions. Blocking the effects of IgE by omalizumab, a recombinant humanized monoclonal antibody that selectively binds to IgE has been shown to be a useful adjunct in the treatment of allergic asthma and rhinitis. Omalizumab is effective as a steroid reducing agent in patients with severe asthma and is successful in decreasing asthma exacerbations. Omalizumab was well tolerated in clinical trials, however, the potential long-term side effects need careful monitoring. The high cost of the molecule could make this a therapeutic option in a small proportion of patients in whom there is a large unmet need.