Hematopoietic stem cell niche generation and maintenance are distinguishable by an epitranscriptomic program.

The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. Whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche is unknown. To address this, we compared perinatal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited enrichment in genes mediating m6A mRNA methylation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs but not osteoblasts led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was significantly rescued by deletion of Klf2, an m6A target. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

A 25-year odyssey of genomic technology advances and structural variant discovery.

This perspective focuses on advances in genome technology over the last 25 years and their impact on germline variant discovery within the field of human genetics. The field has witnessed tremendous technological advances from microarrays to short-read sequencing and now long-read sequencing. Each technology has provided genome-wide access to different classes of human genetic variation. We are now on the verge of comprehensive variant detection of all forms of variation for the first time with a single assay. We predict that this transition will further transform our understanding of human health and biology and, more importantly, provide novel insights into the dynamic mutational processes shaping our genomes.

Automated profiling of gene function during embryonic development.

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients.

Metastatic progression is the main cause of death in cancer patients, whereas the underlying genomic mechanisms driving metastasis remain largely unknown. Here, we assembled MSK-MET, a pan-cancer cohort of over 25,000 patients with metastatic diseases. By analyzing genomic and clinical data from this cohort, we identified associations between genomic alterations and patterns of metastatic dissemination across 50 tumor types. We found that chromosomal instability is strongly correlated with metastatic burden in some tumor types, including prostate adenocarcinoma, lung adenocarcinoma, and HR+/HER2+ breast ductal carcinoma, but not in others, including colorectal cancer and high-grade serous ovarian cancer, where copy-number alteration patterns may be established early in tumor development. We also identified somatic alterations associated with metastatic burden and specific target organs. Our data offer a valuable resource for the investigation of the biological basis for metastatic spread and highlight the complex role of chromosomal instability in cancer progression.

Intranasal pediatric parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in monkeys.

Pediatric SARS-CoV-2 vaccines are needed that elicit immunity directly in the airways as well as systemically. Building on pediatric parainfluenza virus vaccines in clinical development, we generated a live-attenuated parainfluenza-virus-vectored vaccine candidate expressing SARS-CoV-2 prefusion-stabilized spike (S) protein (B/HPIV3/S-6P) and evaluated its immunogenicity and protective efficacy in rhesus macaques. A single intranasal/intratracheal dose of B/HPIV3/S-6P induced strong S-specific airway mucosal immunoglobulin A (IgA) and IgG responses. High levels of S-specific antibodies were also induced in serum, which efficiently neutralized SARS-CoV-2 variants of concern of alpha, beta, and delta lineages, while their ability to neutralize Omicron sub-lineages was lower. Furthermore, B/HPIV3/S-6P induced robust systemic and pulmonary S-specific CD4+ and CD8+ T cell responses, including tissue-resident memory cells in the lungs. Following challenge, SARS-CoV-2 replication was undetectable in airways and lung tissues of immunized macaques. B/HPIV3/S-6P will be evaluated clinically as pediatric intranasal SARS-CoV-2/parainfluenza virus type 3 vaccine.

Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue.

We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-µm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.

Structure and Function of Stator Units of the Bacterial Flagellar Motor.

Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.

Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips.

CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. PAPERCLIP.

Implementing Genome-Driven Oncology.

Early successes in identifying and targeting individual oncogenic drivers, together with the increasing feasibility of sequencing tumor genomes, have brought forth the promise of genome-driven oncology care. As we expand the breadth and depth of genomic analyses, the biological and clinical complexity of its implementation will be unparalleled. Challenges include target credentialing and validation, implementing drug combinations, clinical trial designs, targeting tumor heterogeneity, and deploying technologies beyond DNA sequencing, among others. We review how contemporary approaches are tackling these challenges and will ultimately serve as an engine for biological discovery and increase our insight into cancer and its treatment.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

It's a DoG-eat-DoG world-altered transcriptional mechanisms drive downstream-of-gene (DoG) transcript production.

The past decade has revolutionized our understanding of regulatory noncoding RNAs (ncRNAs). Among the most recently identified ncRNAs are downstream-of-gene (DoG)-containing transcripts that are produced by widespread transcriptional readthrough. The discovery of DoGs has set the stage for future studies to address many unanswered questions regarding the mechanisms that promote readthrough transcription, RNA processing, and the cellular functions of the unique transcripts. In this review, we summarize current findings regarding the biogenesis, function, and mechanisms regulating this exciting new class of RNA molecules.

Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.

The roles of calcium and ATP in the physiology and pathology of the exocrine pancreas.

This review deals with the roles of calcium ions and ATP in the control of the normal functions of the different cell types in the exocrine pancreas as well as the roles of these molecules in the pathophysiology of acute pancreatitis. Repetitive rises in the local cytosolic calcium ion concentration in the apical part of the acinar cells not only activate exocytosis but also, via an increase in the intramitochondrial calcium ion concentration, stimulate the ATP formation that is needed to fuel the energy-requiring secretion process. However, intracellular calcium overload, resulting in a global sustained elevation of the cytosolic calcium ion concentration, has the opposite effect of decreasing mitochondrial ATP production, and this initiates processes that lead to necrosis. In the last few years it has become possible to image calcium signaling events simultaneously in acinar, stellate, and immune cells in intact lobules of the exocrine pancreas. This has disclosed processes by which these cells interact with each other, particularly in relation to the initiation and development of acute pancreatitis. By unraveling the molecular mechanisms underlying this disease, several promising therapeutic intervention sites have been identified. This provides hope that we may soon be able to effectively treat this often fatal disease.

Longitudinal Analysis Reveals Early Development of Three MPER-Directed Neutralizing Antibody Lineages from an HIV-1-Infected Individual.

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

Human Fetal TNF-α-Cytokine-Producing CD4+ Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life.

Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α)+CD4+CD69+ T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4+ T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4+ T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4+ Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4+ T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth.

Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.

DNA single-strand breaks (SSBs) are among the most common lesions in the genome, arising spontaneously and as intermediates of many DNA transactions. Nevertheless, in contrast to double-strand breaks (DSBs), their distribution in the genome has hardly been addressed in a meaningful way. We now present a technique based on genome-wide ligation of 3'-OH ends followed by sequencing (GLOE-Seq) and an associated computational pipeline designed for capturing SSBs but versatile enough to be applied to any lesion convertible into a free 3'-OH terminus. We demonstrate its applicability to mapping of Okazaki fragments without prior size selection and provide insight into the relative contributions of DNA ligase 1 and ligase 3 to Okazaki fragment maturation in human cells. In addition, our analysis reveals biases and asymmetries in the distribution of spontaneous SSBs in yeast and human chromatin, distinct from the patterns of DSBs.

Genomic Answers for Kids: Toward more equitable access to genomic testing for rare diseases in rural populations.

Next-generation sequencing has revolutionized the speed of rare disease (RD) diagnoses. While clinical exome and genome sequencing represent an effective tool for many RD diagnoses, there is room to further improve the diagnostic odyssey of many RD patients. One recognizable intervention lies in increasing equitable access to genomic testing. Rural communities represent a significant portion of underserved and underrepresented individuals facing additional barriers to diagnosis and treatment. Primary care providers (PCPs) at local clinics, though sometimes suspicious of a potential benefit of genetic testing for their patients, have significant constraints in pursuing it themselves and rely on referrals to specialists. Yet, these referrals are typically followed by long waitlists and significant delays in clinical assessment, insurance clearance, testing, and initiation of diagnosis-informed care management. Not only is this process time intensive, but it also often requires multiple visits to urban medical centers for which distance may be a significant barrier to rural families. Therefore, providing early, "direct-to-provider" (DTP) local access to unrestrictive genomic testing is likely to help speed up diagnostic times and access to care for RD patients in rural communities. In a pilot study with a PCP clinic in rural Kansas, we observed a minimum 5.5 months shortening of time to diagnosis through the DTP exome sequencing program as compared to rural patients receiving genetic testing through the "traditional" PCP-referral-to-specialist scheme. We share our experience to encourage future partnerships beyond our center. Our efforts represent just one step in fostering greater diversity and equity in genomic studies.