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This column discusses the benefits and limitations of continuous assessment by reporting on the authors' perspectives of ongoing assessment using examples from patron surveys designed to understand user experience in the physical library and to measure whether needs were met throughout the pandemic. This column will be helpful to anyone involved in assessment or who is interested in what factors to consider while carrying out ongoing assessment or using evidence-based data for decision-making. [ABSTRACT FROM AUTHOR]

This article follows up on two previously published studies regarding the incorporation of student feedback amid the COVID-19 pandemic. It builds on the model of embracing user experience, focusing on how library employees and patrons felt about the health safety protocols in place during Fall 2021. Analyzing both surveys from employees and patrons, the findings indicated that both groups felt safe in the library. This article recommends that when library decision-makers are in doubt about policies and practices, they should consult with the groups directly affected, and demonstrates the importance of ensuring stakeholders feel heard and can contribute to decision-making. [ABSTRACT FROM AUTHOR]

This article demonstrates how a public research university library responded to user needs following radical service changes during Fall 2020 and assesses whether the library met challenges resulting from COVID-19. The library reduced hours and occupancy and implemented a reservation system and new health safety guidelines with the goal of a safe environment. During Fall 2020, 540 survey respondents reported feeling their health was not at risk, suggesting the goal was accomplished. Additionally, users provided feedback about the altered landscape. This article will benefit administrators and user experience librarians who need to balance user preferences and administrative reality. [ABSTRACT FROM AUTHOR]

The University of Illinois Chicago (UIC) Library began planning for the Fall 2020 semester knowing that COVID-19 and social unrest stemming from police actions across the country would impact library safety for the university community. The goal for reopening was to implement best practices that incorporated University health and safety guidelines as well as student feedback. A task force conducted 12 focus group sessions between June 18–26, 2020, in which 56 university students participated. Students identified physical distancing, personal protective equipment (PPE), and monitoring compliance in the library as primary concerns. The campus libraries made extensive changes to its facilities, access to materials and services, and conduct policies to address these issues. This paper will benefit library leaders and administrators making tough and unprecedented time-sensitive decisions using evidence-based data from stakeholders. [ABSTRACT FROM AUTHOR]

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and harbors many mutations in the spike protein, but the origin of this virus variant remains unclear. We address the role of unusual virus evolution mechanisms such as hypermutation, out-of-frame reading, and recombination. Rather, regular Darwinian evolution, that is, the repeated selection of beneficial spike mutations, seems to have led to the appearance of the grossly altered spike protein of the Omicron variant. [ABSTRACT FROM AUTHOR]

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy. [Display omitted] • Preferred transduction of cells expressing two surface markers by bispecific AAVs. • Incorporation of two DARPins without detectable structural changes in the capsid. • Increased uptake rates of bispecific versus monospecific AAVs. • New options for precise in vivo gene delivery to therapy-relevant cells. • Targeting of the HIV reservoir as potential application. [ABSTRACT FROM AUTHOR]

Tools based on RNA interference (RNAi) and the recently developed clustered regularly short palindromic repeats (CRISPR) system enable the selective modification of gene expression, which also makes them attractive therapeutic reagents for combating HIV infection and other infectious diseases. Several parallels can be drawn between the RNAi and CRISPR-Cas9 platforms. An ideal RNAi or CRISPR-Cas9 therapeutic strategy for treating infectious or genetic diseases should exhibit potency, high specificity and safety. However, therapeutic applications of RNAi and CRISPR-Cas9 have been challenged by several major limitations, some of which can be overcome by optimal design of the therapy or the design of improved reagents. In this review, we will discuss some advantages and limitations of anti-HIV strategies based on RNAi and CRISPR-Cas9 with a focus on the efficiency, specificity, off-target effects and delivery methods. [ABSTRACT FROM AUTHOR]

After 40 years under one manager, the Circulation Department at the Richard J. Daley Library was long past due for a change. The challenge of reorganizing included interesting and interrelated aspects: changes to workflows and assignments, moving staff and functions inside the department and across departments, and a deep dive into the culture to which staff had become habituated. Managers eliminated redundancies and increased services, effectiveness, productivity, and joy. This case study is a testament to how successful change happens with patience, respect, and a willingness to be flexible. [ABSTRACT FROM AUTHOR]

Allogeneic transplantation of blood stem cells from a CCR5-Δ32 homozygous donor to an HIV-infected individual, the “Berlin patient”, led to a cure. Since then there has been a search for approaches that mimic this intervention in a gene therapy setting. RNA interference (RNAi) has evolved as a powerful tool to regulate gene expression in a sequence-specific manner and can be used to inactivate the CCR5 mRNA. Short hairpin RNA (shRNA) molecules can impair CCR5 expression, but these molecules may cause unintended side effects and they will not be processed in cells that lack Dicer, such as monocytes. Dicer-independent RNAi pathways have opened opportunities for new AgoshRNA designs that rely exclusively on Ago2 for maturation. Furthermore, AgoshRNA processing yields a single active guide RNA, thus reducing off-target effects. In this study, we tested different AgoshRNA designs against CCR5. We selected AgoshRNAs that potently downregulated CCR5 expression on human T cells and peripheral blood mononuclear cells (PBMC) and that had no apparent adverse effect on T cell development as assessed in a competitive cell growth assay. CCR5 knockdown significantly protected T cells from CCR5 tropic HIV-1 infection. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) infection can be effectively controlled by potent antiviral drugs, but this never results in a cure. The patient should therefore take these drugs for the rest of his/her life, which can cause drug-resistance and adverse effects. Therefore, more durable therapeutic strategies should be considered, such as a stable gene therapy to protect the target T cells against HIV-1 infection. The development of potent therapeutic regimens based on the RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) mechanisms will be described, which can be delivered by lentiviral vectors. These mechanisms attack different forms of the viral genome, the RNA and DNA, respectively, but both mechanisms act in a strictly sequence-specific manner. Early RNAi experiments demonstrated profound virus inhibition, but also indicated that viral escape is possible. Such therapy failure can be prevented by the design of a combinatorial RNAi attack on the virus and this gene therapy is currently being tested in a preclinical humanized mouse model. Recent CRISPR-Cas studies also document robust virus inhibition, but suggest a novel viral escape route that is induced by the cellular nonhomologous end joining DNA repair pathway, which is activated by CRISPR-Cas-induced DNA breaks. We will compare these two approaches for durable HIV-1 suppression and discuss the respective advantages and disadvantages. The potential for future clinical applications will be described. [ABSTRACT FROM AUTHOR]

Bone marrow gene therapy remains an attractive option for treating chronic immunological diseases, including acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV). This technology combines the differentiation and expansion capacity of hematopoietic stem cells (HSCs) with long-term expression of therapeutic transgenes using integrating vectors. In this review we summarize the potential of bone marrow gene therapy for the treatment of HIV/AIDS. A broad range of antiviral strategies are discussed, with a particular focus on RNA-based therapies. The idea is to develop a durable gene therapy that lasts the life span of the infected individual, thus contrasting with daily drug regimens to suppress the virus. Different approaches have been proposed to target either the virus or cellular genes encoding co-factors that support virus replication. Some of these therapies have been tested in clinical trials, providing proof of principle that gene therapy is a safe option for treating HIV/AIDS. In this review several topics are discussed, ranging from the selection of the antiviral molecule and the viral target to the optimal vector system for gene delivery and the setup of appropriate preclinical test systems. The molecular mechanisms used to formulate a cure for HIV infection are described, including the latest antiviral strategies and their therapeutic applications. Finally, a potent combination of anti-HIV genes based on our own research program is described. [ABSTRACT FROM AUTHOR]

Highlights: [•] The use of T cell lines to replace PBMCS for HIV-1 studies has been studied. [•] The PM1 T cell line supports the replication of all HIV-1 subtypes. [•] The PM1 T cell line supports CCR5- and CXCR4-using HIV-1 variants. [•] The safety and efficacy of an RNAi-based anti-HIV gene therapy were evaluated. [•] The PM1 cells provide a valuable tool for basic and applied HIV-1 studies. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is highly effective in inhibiting human immunodeficiency virus type 1 (HIV-1) replication by the expression of antiviral short hairpin RNA (shRNA) in stably transduced T-cell lines. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against highly conserved regions of the HIV-1 RNA genome. The future in vivo application of such a gene therapy protocol will reach only a fraction of the T cells, such that HIV-1 replication will continue in the unmodified T cells, thereby possibly frustrating the therapy by generation of HIV-1 variants that escape from the inhibition imposed by the protected cells. We studied virus inhibition and evolution in pure cultures of shRNA-expressing cells versus mixed cell cultures of protected and unprotected T cells. The addition of the unprotected T cells indeed seems to accelerate HIV-1 evolution and escape from a single shRNA inhibitor. However, expression of three antiviral shRNAs from a single lentiviral vector prevents virus escape even in the presence of unprotected cells. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models. [ABSTRACT FROM AUTHOR]

Background: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. Methods: Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. Results: Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. Conclusions: These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits. [ABSTRACT FROM AUTHOR]

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders crossreacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir. [ABSTRACT FROM AUTHOR]

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus −RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure. [ABSTRACT FROM AUTHOR]

Coronavirus disease 19 (COVID-19) patients usually require long periods of mechanical ventilation and sedation, which added to steroid therapy, favours a predisposition to the development of delirium and subsequent mental health disorders, as well as physical and respiratory sequelae. The aim of this study was to determine the prevalence of post-intensive care syndrome (PICS) at 3 months after hospital discharge, in a cohort of mechanically ventilated patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An ambispective, observational study was conducted in three hospitals with intensive care unit (ICU) follow-up clinics. We studied adults who survived a critical illness due to SARS-CoV-2 infection requiring invasive mechanical ventilation. A physical (muscle strength and pulmonary function), functional [12-Item Short Form Health Survey (SF-12), and Barthel score], psychological [hospital anxiety and depression (HADS) and posttraumatic stress disorder symptom severity scales], and cognitive [Montreal cognitive assessment (MoCA) test] assessment were performed. A total of 186 patients were evaluated at 88 days (IQR 68–121) after hospital discharge. Mean age was 59 ± 12 years old, 126 (68%) patients were men, and median length of mechanical ventilation was 14 days (IQR 8–31). About 3 out of 4 patients (n = 139, 75%) met PICS criteria. Symptoms of cognitive and psychiatric disorders were found in 59 (32%) and 58 (31%) patients, respectively. Ninety-one (49%) patients had muscle weakness. Pulmonary function tests in patients with no respiratory comorbidities showed a normal pattern in 93 (50%) patients, and a restrictive disorder in 62 (33%) patients. Also, 69 patients (37%) were on sick leave, while 32 (17%) had resumed work at the time of assessment. In conclusion, survivors of critical illness due to SARS-CoV-2 infection requiring mechanical ventilation have a high prevalence of PICS. Physical domain is the most frequently damaged, followed by cognitive and psychiatric disorders. ICU follow-up clinics enable the assistance of this vulnerable population. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) can be induced by intracellular expression of a short hairpin RNA (shRNA). Processing of the shRNA requires the RNaseIII-like Dicer enzyme to remove the loop and to release the biologically active small interfering RNA (siRNA). Dicer is also involved in microRNA (miRNA) processing to liberate the mature miRNA duplex, but recent studies indicate that miR-451 is not processed by Dicer. Instead, this miRNA is processed by the Argonaute 2 (Ago2) protein, which also executes the subsequent cleavage of a complementary mRNA target. Interestingly, shRNAs that structurally resemble miR-451 can also be processed by Ago2 instead of Dicer. The key determinant of these “AgoshRNA” molecules is a relatively short basepaired stem, which avoids Dicer recognition and consequently allows alternative processing by Ago2. AgoshRNA processing yields a single active RNA strand, whereas standard shRNAs produce a duplex with guide and passenger strands and the latter may cause adverse off-target effects. In this study, we converted previously tested active anti-HIV-1 shRNA molecules into AgoshRNA. We tested several designs that could potentially improve AgoshRNA activity, including extension of the complementarity between the guide strand and the mRNA target and reduction of the thermodynamic stability of the hairpins. We demonstrate that active AgoshRNAs can be generated. However, the RNAi activity is reduced compared to the matching shRNAs. Despite reduced RNAi activity, comparison of an active AgoshRNA and the matching shRNA in a sensitive cell toxicity assay revealed that the AgoshRNA is much less toxic. [ABSTRACT FROM AUTHOR]

RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both). [ABSTRACT FROM AUTHOR]

The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a. We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells. We demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such "hypermutated" HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation. These results provide in vitro "proof of concept' for CRISPR-Cas12b mediated HIV-1 inactivation. [Display omitted] • CRISPR-Cas12b-based strategies that result in inactivation of integrated HIV genomes hold promise as a potential cure for HIV. • Cas12b can achieve full HIV inactivation with only a single gRNA. • Combination of two antiviral gRNAs led to improved anti-HIV potency and more grossly mutated HIV proviruses. [ABSTRACT FROM AUTHOR]

El objetivo de este estudio es analizar la relación entre la gestión del conocimiento y el crecimiento, utilizando una muestra de 217 empresas del sector manufacturero de Aguascalientes (México). El trabajo de campo de la investigación se realizó durante el periodo Enero-Abril del año 2017, utilizando como método de recolección de la información el método de encuesta a través de la aplicación de un cuestionario. Con los resultados obtenidos se concluyó que la gestión del conocimiento tiene efectos positivos y significativos en el crecimiento de las empresas de la industria manufacturera de Aguascalientes. [ABSTRACT FROM AUTHOR]

Type 3 RNA polymerase III (Pol III) promoters are widely used for the expression of small RNAs such as short hairpin RNA and guide RNA in the popular RNAi and CRISPR-Cas gene regulation systems. Although it is generally believed that type 3 Pol III promoters use a defined transcription start site (+1 position), most man-made promoter constructs contain local sequence alterations of which the impact on transcription efficiency and initiation accuracy is not known. For three human type 3 Pol III promoters (7SK, U6, and H1), we demonstrated that the nucleotides around the +1 position affect both the transcriptional efficiency and start site selection. Human 7SK and U6 promoters with A or G at the +1 position efficiently produced small RNAs with a precise +1 start site. The human H1 promoter with +1A or G also efficiently produced small RNAs but from multiple start sites in the −3/−1 window. These results provide new insights for the design of vectors for accurate expression of designed small RNAs for research and therapeutic purposes. [ABSTRACT FROM PUBLISHER]

Although hepatitis E has emerged as a global health issue, there is limited knowledge of its infection biology and no FDA-approved medication is available. Aiming to investigate the role of protein kinases in hepatitis E virus (HEV) infection and to identify potential antiviral targets, we screened a library of pharmacological kinase inhibitors in a cell culture model, a subgenomic HEV replicon containing luciferase reporter. We identified protein kinase C alpha (PKCα) as an essential cell host factor restricting HEV replication. Both specific inhibitor and shRNA-mediated knockdown of PKCα enhanced HEV replication. Conversely, over-expression of the activated form of PKCα or treatment with its pharmacological activator strongly inhibited HEV replication. Interestingly, upon the stimulation by its activator, PKCα efficiently activates its downstream Activator Protein 1 (AP-1) pathway, leading to the induction of antiviral interferon-stimulated genes (ISGs). This process is independent of the JAK-STAT machinery and interferon production. However, PKCα induced HEV inhibition appears independent of the AP1 cascade. The discovery that activated PKCα restricts HEV replication reveals new insight of HEV-host interactions and provides new target for antiviral drug development. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs. [ABSTRACT FROM AUTHOR]