High-resolution Inference of Multiplexed Anti-HIV Gene Editing using Single-Cell Targeted DNA Sequencing.

Gene therapy-based HIV cure strategies typically aim to excise the HIV provirus directly, or target host dependency factors (HDFs) that support viral persistence. Cure approaches will likely require simultaneous co-targeting of multiple sites within the HIV genome to prevent evolution of resistance, and/or co-targeting of multiple HDFs to fully render host cells refractory to HIV infection. Bulk cell-based methods do not enable inference of co-editing within individual viral or target cell genomes, and do not discriminate between monoallelic and biallelic gene disruption. Here, we describe a targeted single-cell DNA sequencing (scDNA-seq) platform characterizing the near full-length HIV genome and 50 established HDF genes, designed to evaluate anti-HIV gene therapy strategies. We implemented the platform to investigate the capacity of multiplexed CRISPR-Cas9 ribonucleoprotein complexes (Cas9-RNPs) to simultaneously 1) inactivate the HIV provirus, and 2) knockout the CCR5 and CXCR4 HDF (entry co-receptor) genes in microglia and primary monocyte-derived macrophages (MDMs). Our scDNA-seq pipeline revealed that antiviral gene editing is rarely observed at multiple loci (or both alleles of a locus) within an individual cell, and editing probabilities across sites are linked. Our results demonstrate that single-cell sequencing is critical to evaluate the true efficacy and therapeutic potential of HIV gene therapy.

Systematic benchmarking of single-cell ATAC-sequencing protocols.

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.

Human galectin-9 potently enhances SARS-CoV-2 replication and inflammation in airway epithelial cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global economic and health crisis. Recently, plasma levels of galectin-9 (Gal-9), a ß-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis, were reported to be elevated in the setting of severe COVID-19 disease. However, the impact of Gal-9 on SARS-CoV-2 infection and immunopathology remained to be elucidated. In this study, we demonstrate that Gal-9 treatment potently enhances SARS-CoV-2 replication in human airway epithelial cells (AECs), including immortalized AECs and primary AECs cultured at the air-liquid interface. Gal-9-glycan interactions promote SARS-CoV-2 attachment and entry into AECs in an angiotensin-converting enzyme 2 (ACE2)-dependent manner, enhancing the binding of the viral spike protein to ACE2. Transcriptomic analysis revealed that Gal-9 and SARS-CoV-2 infection synergistically induced the expression of key pro-inflammatory programs in AECs, including the IL-6, IL-8, IL-17, EIF2, and TNFα signaling pathways. Our findings suggest that manipulation of Gal-9 should be explored as a therapeutic strategy for SARS-CoV-2 infection.

Human Galectin-9 Potently Enhances SARS-CoV-2 Replication and Inflammation in Airway Epithelial Cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global economic and health crisis. Recently, plasma levels of galectin-9 (Gal-9), a ß-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis, were reported to be elevated in the setting of severe COVID-19 disease. However, the impact of Gal-9 on SARS-CoV-2 infection and immunopathology remained to be elucidated. Here, we demonstrate that Gal-9 treatment potently enhances SARS-CoV-2 replication in human airway epithelial cells (AECs), including primary AECs in air-liquid interface (ALI) culture. Gal-9-glycan interactions promote SARS-CoV-2 attachment and entry into AECs in an ACE2-dependent manner, enhancing the binding affinity of the viral spike protein to ACE2. Transcriptomic analysis revealed that Gal-9 and SARS-CoV-2 infection synergistically induce the expression of key pro-inflammatory programs in AECs including the IL-6, IL-8, IL-17, EIF2, and TNFα signaling pathways. Our findings suggest that manipulation of Gal-9 should be explored as a therapeutic strategy for SARS-CoV-2 infection. Importance: COVID-19 continues to have a major global health and economic impact. Identifying host molecular determinants that modulate SARS-CoV-2 infectivity and pathology is a key step in discovering novel therapeutic approaches for COVID-19. Several recent studies have revealed that plasma concentrations of the human ß-galactoside-binding protein galectin-9 (Gal-9) are highly elevated in COVID-19 patients. In this study, we investigated the impact of Gal-9 on SARS-CoV-2 pathogenesis ex vivo in airway epithelial cells (AECs), the critical initial targets of SARS-CoV-2 infection. Our findings reveal that Gal-9 potently enhances SARS-CoV-2 replication in AECs, interacting with glycans to enhance the binding between viral particles and entry receptors on the target cell surface. Moreover, we determined that Gal-9 accelerates and exacerbates several virus-induced pro-inflammatory programs in AECs that are established signature characteristics of COVID-19 disease and SARS-CoV-2-induced acute respiratory distress syndrome (ARDS). Our findings suggest that Gal-9 is a promising pharmacological target for COVID-19 therapies.

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.

Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes, and the hybrid promoters can be induced using estradiol, a compound with no detectable impact on S. cerevisiae physiology. Using combinations of one, two or three operator sequence repeats and a set of native S. cerevisiae promoters, we obtained a series of hybrid promoters that can be induced to different levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain.

Engineering microbial biofuel tolerance and export using efflux pumps.

Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade-off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n-butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes.

Structure of the C-terminus of the mRNA export factor Dbp5 reveals the interaction surface for the ATPase activator Gle1.

The DExD/H-box RNA-dependent ATPase Dbp5 plays an essential role in the nuclear export of mRNA. Dbp5 localizes to the nuclear pore complex, where its ATPase activity is stimulated by Gle1 and its coactivator inositol hexakisphosphate. Here, we present the crystal structure of the C-terminal domain of Dbp5, refined to 1.8 A. The structure reveals a RecA-like fold that contains two defining characteristics not present in other structurally characterized DExD/H-box proteins: a C-terminal alpha-helix and a loop connecting beta5 and alpha4, both of which are composed of conserved and unique elements in the Dbp5 primary sequence. Using structure-guided mutagenesis, we have identified several charged surface residues that, when mutated, weaken the binding of Gle1 and inhibit the ability of Gle1 to stimulate Dbp5's ATPase activity. In vivo analysis of the same mutations reveals that those mutants displaying the weakest ATPase stimulation in vitro are also unable to support yeast growth. Analysis of the correlation between the in vitro and in vivo data indicates that a threshold level of Dbp5 ATPase activity is required for cellular mRNA export that is not met by the unstimulated enzyme, suggesting a possible mechanism by which Dbp5's activity can be modulated to regulate mRNA export.