CRISPR activation screens: navigating technologies and applications.

Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery is continually under development to achieve greater, more robust, and more consistent activation. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening.

Advanced pathophysiology mimicking lung models for accelerated drug discovery.

BACKGROUND: Respiratory diseases are the 2nd leading cause of death globally. The current treatments for chronic lung diseases are only supportive. Very few new classes of therapeutics have been introduced for lung diseases in the last 40 years, due to the lack of reliable lung models that enable rapid, cost-effective, and high-throughput testing. To accelerate the development of new therapeutics for lung diseases, we established two classes of lung-mimicking models: (i) healthy, and (ii) diseased lungs - COPD. METHODS: To establish models that mimic the lung complexity to different extents, we used five design components: (i) cell type, (ii) membrane structure/constitution, (iii) environmental conditions, (iv) cellular arrangement, (v) substrate, matrix structure and composition. To determine whether the lung models are reproducible and reliable, we developed a quality control (QC) strategy, which integrated the real-time and end-point quantitative and qualitative measurements of cellular barrier function, permeability, tight junctions, tissue structure, tissue composition, and cytokine secretion. RESULTS: The healthy model is characterised by (i) continuous tight junctions, (ii) physiological cellular barrier function, (iii) a full thickness epithelium composed of multiple cell layers, and (iv) the presence of ciliated cells and goblet cells. Meanwhile, the disease model emulates human COPD disease: (i) dysfunctional cellular barrier function, (ii) depletion of ciliated cells, and (ii) overproduction of goblet cells. The models developed here have multiple competitive advantages when compared with existing in vitro lung models: (i) the macroscale enables multimodal and correlative characterisation of the same model system, (ii) the use of cells derived from patients that enables the creation of individual models for each patient for personalised medicine, (iii) the use of an extracellular matrix proteins interface, which promotes physiological cell adhesion and differentiation, (iv) media microcirculation that mimics the dynamic conditions in human lungs. CONCLUSION: Our model can be utilised to test safety, efficacy, and superiority of new therapeutics as well as to test toxicity and injury induced by inhaled pollution or pathogens. It is envisaged that these models can also be used to test the protective function of new therapeutics for high-risk patients or workers exposed to occupational hazards.

Fibroblast-expressed LRRC15 is a receptor for SARS-CoV-2 spike and controls antiviral and antifibrotic transcriptional programs.

Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

The VEGAS Platform Is Unsuitable for Mammalian Directed Evolution.

Directed evolution uses cycles of gene diversification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or "VEGAS") has been described. Here we report that the VEGAS system has major limitations that preclude its use for directed evolution. The deconstructed Sindbis virus (SINV) genome that comprises the VEGAS system could no longer promote Sindbis structural gene (SSG)-dependent viral replication. Moreover, viral particles generated using the VEGAS system rapidly lost the target directed evolution transgene, and instead, "cheater" particles, primarily containing RNA encoding SINV structural components, arose. By sequencing, we found that this contamination came from RNA provided during initial SINV packaging, not RNA derived from the VEGAS system. Of note, both the structural RNA and target transgenes used in the VEGAS system contain viral packaging sequences. The impact of SINV "cheater" particles could be potentially overcome in the context of a robust VEGAS circuit, but since SSG complementation is also defective in the VEGAS system, selection for authentic evolution products is not currently possible. Similar results have been obtained in independent laboratories. Taken together, these results show that the VEGAS system does not work as described and, without significant redesign, cannot be used for mammalian directed evolution campaigns.

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing.

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

A putative WAVE regulatory complex (WRC) interacting receptor sequence (WIRS) in the cytoplasmic tail of HSV-1 gE does not function in WRC recruitment or neuronal transport.

HSV-1 envelope glycoprotein E (gE) is important for viral egress and cell-to-cell spread but the host protein(s) involved in these functions have yet to be determined. We aimed to investigate a role for the Arp2/3 complex and actin regulation in viral egress based on the identification of a WAVE Regulatory Complex (WRC) Interacting Receptor Sequence (WIRS) in the cytoplasmic tail (CT) of gE. A WIRS-dependent interaction between the gE(CT) and subunits of the WRC was demonstrated by GST-pulldown assay and a role for the Arp2/3 complex in cell-to-cell spread was also observed by plaque assay. Subsequent study of a recombinant HSV-1 gE WIRS-mutant found no significant changes to viral production and release based on growth kinetics studies, or changes to plaque and comet size in various cell types, suggesting no function for the motif in cell-to-cell spread. GFP-Trap pulldown and proximity ligation assays were unable to confirm a WIRS-dependent interaction between gE and the WRC in human cell lines though the WIRS-independent interaction observed in situ warrants further study. Confocal microscopy of infected cells of neuronal origin identified no impairment of gE WIRS-mutant HSV-1 anterograde transport along axons. We propose that the identified gE WIRS motif does not function directly in recruitment of the WRC in human cells, in cell-to-cell spread of virus or in anterograde transport along axons. Further studies are needed to understand how HSV-1 manipulates and traverses the actin cytoskeleton and how gE may contribute to these processes in a WIRS-independent manner.

A short overview of CRISPR-Cas technology and its application in viral disease control.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) together with CRISPR-associated (Cas) proteins have catalysed a revolution in genetic engineering. Native CRISPR-Cas systems exist in many bacteria and archaea where they provide an adaptive immune response through sequence-specific degradation of an invading pathogen's genome. This system has been reconfigured for use in genome editing, drug development, gene expression regulation, diagnostics, the prevention and treatment of cancers, and the treatment of genetic and infectious diseases. In recent years, CRISPR-Cas systems have been used in the diagnosis and control of viral diseases, for example, CRISPR-Cas12/13 coupled with new amplification techniques to improve the specificity of sequence-specific fluorescent probe detection. Importantly, CRISPR applications are both sensitive and specific and usually only require commonly available lab equipment. Unlike the canonical Cas9 which is guided to double-stranded DNA sites of interest, Cas13 systems target RNA sequences and thus can be employed in strategies directed against RNA viruses or for transcriptional silencing. Many challenges remain for these approach, including issues with specificity and the requirement for better mammalian delivery systems. In this review, we summarize the applications of CRISPR-Cas systems in controlling mammalian viral infections. Following necessary improvements, it is expected that CRISPR-Cas systems will be used effectively for such applications in the future.

Tour de Herpes: Cycling Through the Life and Biology of HSV-1.

Herpes simplex virus type 1 (HSV-1) is a prevalent and important human pathogen that has been studied in a wide variety of contexts. This book provides protocols currently in use in leading laboratories in many fields of HSV-1 research. This introductory chapter gives a brief overview of HSV-1 biology and life cycle, covering basic aspects of virus structure, the prevalence of and diseases caused by the virus, replication in cultured cells, viral latency, antiviral defenses, and the mechanisms that the virus uses to counteract these defenses.

Infection and Transport of Herpes Simplex Virus Type 1 in Neurons: Role of the Cytoskeleton.

Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that has the ability to infect and replicate within epithelial cells and neurons and establish a life-long latent infection in sensory neurons. HSV-1 depends on the host cellular cytoskeleton for entry, replication, and exit. Therefore, HSV-1 has adapted mechanisms to promote its survival by exploiting the microtubule and actin cytoskeletons to direct its active transport, infection, and spread between neurons and epithelial cells during primary and recurrent infections. This review will focus on the currently known mechanisms utilized by HSV-1 to harness the neuronal cytoskeleton, molecular motors, and the secretory and exocytic pathways for efficient virus entry, axonal transport, replication, assembly, and exit from the distinct functional compartments (cell body and axon) of the highly polarized sensory neurons.

Cytoskeletons in the Closet-Subversion in Alphaherpesvirus Infections.

Actin filaments, microtubules and intermediate filaments form the cytoskeleton of vertebrate cells. Involved in maintaining cell integrity and structure, facilitating cargo and vesicle transport, remodelling surface structures and motility, the cytoskeleton is necessary for the successful life of a cell. Because of the broad range of functions these filaments are involved in, they are common targets for viral pathogens, including the alphaherpesviruses. Human-tropic alphaherpesviruses are prevalent pathogens carried by more than half of the world's population; comprising herpes simplex virus (types 1 and 2) and varicella-zoster virus, these viruses are characterised by their ability to establish latency in sensory neurons. This review will discuss the known mechanisms involved in subversion of and transport via the cytoskeleton during alphaherpesvirus infections, focusing on protein-protein interactions and pathways that have recently been identified. Studies on related alphaherpesviruses whose primary host is not human, along with comparisons to more distantly related beta and gammaherpesviruses, are also presented in this review. The need to decipher as-yet-unknown mechanisms exploited by viruses to hijack cytoskeletal components-to reveal the hidden cytoskeletons in the closet-will also be addressed.