High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma.

INTRODUCTION: Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterized by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. We aimed to identify genetic and morphological variation that can be attributed to trabecular meshwork cell (TMC) dysfunction and raised IOP in POAG. METHODS: 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescence coupled with CellProfiler image analysis allowed for single-cell morphological profiling. RESULTS: Many gene knockouts invoked DEGs relating to matrix metalloproteinases and interferon-induced proteins. We have prioritized genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified, suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, while knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSION: High-throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

DNA-Nanostructure-Guided Assembly of Proteins into Programmable Shapes.

The development of methods to synthesize artificial protein complexes with precisely controlled configurations will enable diverse biological and medical applications. Using DNA to link proteins provides programmability that can be difficult to achieve with other methods. Here, we use DNA origami as an "assembler" to guide the linking of protein-DNA conjugates using a series of oligonucleotide hybridization and displacement operations. We constructed several isomeric protein nanostructures, including a dimer, two types of trimer structures, and three types of tetramer assemblies, on a DNA origami platform by using a C3-symmetric building block composed of a protein trimer modified with DNA handles. Our approach expands the scope for the precise assembly of protein-based nanostructures and will enable the formulation of functional protein complexes with stoichiometric and geometric control.

Structure-Dependent Electrical Conductance of DNA Origami Nanowires.

Exploring the structural and electrical properties of DNA origami nanowires is an important endeavor for the advancement of DNA nanotechnology and DNA nanoelectronics. Highly conductive DNA origami nanowires are a desirable target for creating low-cost self-assembled nanoelectronic devices and circuits. In this work, the structure-dependent electrical conductance of DNA origami nanowires is investigated. A silicon nitride (Si3 N4 ) on silicon semiconductor chip with gold electrodes was used for collecting electrical conductance measurements of DNA origami nanowires, which are found to be an order of magnitude less electrically resistive on Si3 N4 substrates treated with a monolayer of hexamethyldisilazane (HMDS) (∼1013 ohms) than on native Si3 N4 substrates without HMDS (∼1014 ohms). Atomic force microscopy (AFM) measurements of the height of DNA origami nanowires on mica and Si3 N4 substrates reveal that DNA origami nanowires are ∼1.6 nm taller on HMDS-treated substrates than on the untreated ones indicating that the DNA origami nanowires undergo increased structural deformation when deposited onto untreated substrates, causing a decrease in electrical conductivity. This study highlights the importance of understanding and controlling the interface conditions that affect the structure of DNA and thereby affect the electrical conductance of DNA origami nanowires.

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures.

DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site-specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to "smart" functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.

Programmable Site-Specific Functionalization of DNA Origami with Polynucleotide Brushes.

Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush-functionalized DNA nanostructures. We demonstrate that SI-TcEP can site-specifically pattern DONs with brushes containing both natural and non-natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse-grained simulations predict the conformation of the brush-functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush-functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site-specific growth of polynucleotide brushes. The ability to site-specifically decorate DONs with brushes of natural and non-natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems.

Deep Learning-Based Identification of Intraocular Pressure-Associated Genes Influencing Trabecular Meshwork Cell Morphology.

Purpose: Genome-wide association studies have recently uncovered many loci associated with variation in intraocular pressure (IOP). Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs) and thus, IOP regulation. Design: Experimental study. Subjects: Primary TMCs collected from human donors. Methods: Sixty-two genes at 55 loci associated with IOP variation were knocked out in primary TMC lines. All cells underwent high-throughput microscopy imaging after being stained with a 5-channel fluorescent cell staining protocol. A convolutional neural network was trained to distinguish between gene knockout and normal control cell images. The area under the receiver operator curve (AUC) metric was used to quantify morphological variation in gene knockouts to identify potential pathological perturbations. Main Outcome Measures: Degree of morphological variation as measured by deep learning algorithm accuracy of differentiation from normal controls. Results: Cells where LTBP2 or BCAS3 had been perturbed demonstrated the greatest morphological variation from normal TMCs (AUC 0.851, standard deviation [SD] 0.030; and AUC 0.845, SD 0.020, respectively). Of 7 multigene loci, 5 had statistically significant differences in AUC (P < 0.05) between genes, allowing for pathological gene prioritization. The mitochondrial channel most frequently showed the greatest degree of morphological variation (33.9% of cell lines). Conclusions: We demonstrate a robust method for functionally interrogating genome-wide association signals using high-throughput microscopy and AI. Genetic variations inducing marked morphological variation can be readily identified, allowing for the gene-based dissection of loci associated with complex traits. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

New Endothelial Corneal Dystrophy in a Chinese Family.

PURPOSE: The aim of this study was to characterize the clinical presentation of atypical endothelial corneal dystrophy (ECD) and to identify possible associated genetic variants in a Chinese family. METHODS: Six affected members, 4 unaffected first-degree relatives, and 3 spouses who were enrolled in this study underwent ophthalmic examinations. Genetic linkage analysis was performed for 4 affected and 2 unaffected members, and whole-exome sequencing (WES) was performed for 2 patients to identify disease-causing variants. Candidate causal variants were verified using Sanger sequencing in family members and 200 healthy controls. RESULTS: The mean age at disease onset was 16.5 years. The early phenotype of this atypical ECD was characterized by multiple small white translucent spots located in Descemet membrane of the peripheral cornea. These spots coalesced to form opacities with variable shapes, and eventually merged along the limbus. Subsequently, translucent spots appeared in central Descemet membrane and accumulated, causing diffuse polymorphous opacities over time. Finally, significant endothelial decompensation led to diffuse corneal edema. A heterozygous missense variant in the KIAA1522 gene (c.1331G>A; p.R444Q) was identified by WES, which was present in all 6 patients but was absent in the unaffected members and healthy controls. CONCLUSIONS: The clinical features of atypical ECD are unique compared with those of known corneal dystrophies. Moreover, genetic analysis identified the c.1331G>A variant in KIAA1522 , which may be responsible for the pathogenesis of this atypical ECD. Thus, we propose this is a new form of ECD based on our clinical findings.

Analysis of Ocular Surface Characteristics and Incidence of Dry Eye Disease in Systemic Lupus Erythematosus Patients Without Secondary Sjögren's Syndrome.

Objective: To investigate the differences in ocular surface characteristics, tear film quality, and the incidence of dry eye disease (DED) between Systemic Lupus Erythematosus (SLE) patients and healthy populations. Methods: This age and gender-matched cross-sectional study included 96 SLE patients without secondary Sjögren's syndrome (SS) and 72 healthy subjects. The Ocular Surface Disease Index (OSDI), tear meniscus height (TMH), non-invasive tear film breakup time (NIKBUT), meibography, and tear film lipid layer grade were assessed. A receiver operative characteristic (ROC) curve was constructed to evaluate the predictive value of risk factors. Results: Compared with the control subjects, a significantly greater proportion of SLE patients met the TFOS DEWS II DED diagnostic criteria (34.3 vs. 18.1%, P = 0.019). SLE patients without SS had higher OSDI scores [10.0 (4.5,18.0) vs. 5.0 (2.5,11.9), P < 0.001], and shorter NIKBUT [9.6 (6.6,15.0) vs. 12.3 (8.4, 15.8), P = 0.035]. Furthermore, TMH, Tear film lipid layer grade, and Meibomian gland (MG) dropout in SLE patients were worse than those in control subjects (all P < 0.05). For ROC analysis, the area under curve (AUC), sensitivity and specificity of prediction were 0.915, 75.8 and 92.1% for the combination of SLE disease activity index (SLEDAI), age and NIKBUT. Conclusions: SLE patients without SS exhibited a higher risk for DED than healthy subjects, and the poorer Meibomian gland function in SLE patients may potentially contribute to the development of DED. The combined parameters of SLEDAI, age and NIKBUT showed a high efficiency for the diagnosis of DED in SLE patients, with practical clinical applications.

Single-cell eQTL mapping identifies cell type-specific genetic control of autoimmune disease.

The human immune system displays substantial variation between individuals, leading to differences in susceptibility to autoimmune disease. We present single-cell RNA sequencing (scRNA-seq) data from 1,267,758 peripheral blood mononuclear cells from 982 healthy human subjects. For 14 cell types, we identified 26,597 independent cis-expression quantitative trait loci (eQTLs) and 990 trans-eQTLs, with most showing cell type-specific effects on gene expression. We subsequently show how eQTLs have dynamic allelic effects in B cells that are transitioning from naïve to memory states and demonstrate how commonly segregating alleles lead to interindividual variation in immune function. Finally, using a Mendelian randomization approach, we identify the causal route by which 305 risk loci contribute to autoimmune disease at the cellular level. This work brings together genetic epidemiology with scRNA-seq to uncover drivers of interindividual variation in the immune system.

Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing.

CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.