Transcriptional coupling of telomeric retrotransposons with the cell cycle.

Unlike most species that use telomerase for telomere maintenance, many dipterans, including , rely on three telomere-specific retrotransposons (TRs)-, , and -to form tandem repeats at chromosome ends. Although TR transcription is crucial in their life cycle, its regulation remains poorly understood. This study identifies the Mediator complex, E2F1-Dp, and Scalloped/dTEAD as key regulators of TR transcription.

De novo structural variants in autism spectrum disorder disrupt distal regulatory interactions of neuronal genes.

Three-dimensional genome organization plays a critical role in gene regulation, and disruptions can lead to developmental disorders by altering the contact between genes and their distal regulatory elements. Structural variants (SVs) can disturb local genome organization, such as the merging of topologically associating domains upon boundary deletion. Testing large numbers of SVs experimentally for their effects on chromatin structure and gene expression is time and cost prohibitive.

CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription.

Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identified a total of 39 functional enhancers for four dosage-sensitive genes, , , , and , using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. We found that enhancer annotation provides potential pathological insights into disease-associated copy number variants.

Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.

High-throughput PRIME-editing screens identify functional DNA variants in the human genome.

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and single-base resolution manner remains challenging. Here, we develop a pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp MYC enhancer via PRIME-mediated single-base resolution analysis.

Transcriptional coupling of telomeric retrotransposons with the cell cycle.

Instead of employing telomerases to safeguard chromosome ends, dipteran species maintain their telomeres by transposition of telomeric-specific retrotransposons (TRs): in , these are , , and . Previous studies have shown how these TRs create tandem repeats at chromosome ends, but the exact mechanism controlling TR transcription has remained unclear. Here we report the identification of multiple subunits of the transcription cofactor Mediator complex and transcriptional factors Scalloped (Sd, the TEAD homolog in flies) and E2F1-Dp as novel regulators of TR transcription and telomere length in .

Functional characterization of Alzheimer's disease genetic variants in microglia.

Candidate cis-regulatory elements (cCREs) in microglia demonstrate the most substantial enrichment for Alzheimer's disease (AD) heritability compared to other brain cell types. However, whether and how these genome-wide association studies (GWAS) variants contribute to AD remain elusive. Here we prioritize 308 previously unreported AD risk variants at 181 cCREs by integrating genetic information with microglia-specific 3D epigenome annotation.

High throughput PRIME editing screens identify functional DNA variants in the human genome.

Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and base-pair resolution manner remains challenging. Here, we develop a novel pooled prime editing screen method, PRIME, which can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp enhancer via PRIME-mediated saturation mutagenesis.

Efficient bi-allelic tagging in human induced pluripotent stem cells using CRISPR.

Allelic tagging of endogenous genes enables studying gene function and transcriptional control in the native genomic context. Here, we present an efficient protocol for bi-allelic tagging of protein-coding genes with fluorescent reporters in human iPSCs using the CRISPR-Cas9-mediated homology-directed repair. We detail steps for design, cloning, electroporation, and single-cell clone isolation and validation.

Structure Design of Polymer-Based Films for Passive Daytime Radiative Cooling.

Passive daytime radiative cooling (PDRC), a cooling method that needs no additional energy, has become increasingly popular in recent years. The combination of disordered media and polymeric photonics will hopefully lead to the large-scale fabrication of high-performance PDRC devices. This work aims to study two typical PDRC structures, the randomly distributed silica particle (RDSP) structure and the porous structure, and systematically investigates the effects of structural parameters (diameter , volume fraction fv, and thickness ) on the radiative properties of the common plastic materials.

Ultralow Interfacial Thermal Resistance of Graphene Thermal Interface Materials with Surface Metal Liquefaction.

Developing advanced thermal interface materials (TIMs) to bridge heat-generating chip and heat sink for constructing an efficient heat transfer interface is the key technology to solve the thermal management issue of high-power semiconductor devices. Based on the ultra-high basal-plane thermal conductivity, graphene is an ideal candidate for preparing high-performance TIMs, preferably to form a vertically aligned structure so that the basal-plane of graphene is consistent with the heat transfer direction of TIM. However, the actual interfacial heat transfer efficiency of currently reported vertically aligned graphene TIMs is far from satisfactory.

Genetically encoded chemical crosslinking of RNA in vivo.

Protein-RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo.

High-throughput CRISPRi and CRISPRa technologies in 3D genome regulation for neuropsychiatric diseases.

Advances in genomics have led to the identification of many risk loci with hundreds of genes and thousands of DNA variants associated with neuropsychiatric disorders. A significant barrier to understanding the genetic underpinnings of complex diseases is the lack of functional characterization of risk genes and variants in biological systems relevant to human health and connecting disease-associated variants to pathological phenotypes. Characterizing gene and DNA variant functions requires genetic perturbations followed by molecular and cellular assays of neurobiological phenotypes.

Parallel characterization of cis-regulatory elements for multiple genes using CRISPRpath.

Current pooled CRISPR screens for cis-regulatory elements (CREs), based on transcriptional output changes, are typically limited to characterizing CREs of only one gene. Here, we describe CRISPRpath, a scalable screening strategy for parallelly characterizing CREs of genes linked to the same biological pathway and converging phenotypes. We demonstrate the ability of CRISPRpath for simultaneously identifying functional enhancers of six genes in the 6-thioguanine–induced DNA mismatch repair pathway using both CRISPR interference (CRISPRi) and CRISPR nuclease (CRISPRn) approaches.

Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in .

The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence.

The Mediator CDK8-Cyclin C complex modulates Dpp signaling in Drosophila by stimulating Mad-dependent transcription.

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs.

MiR-315 is required for neural development and represses the expression of dFMR1 in Drosophila melanogaster.

Aim: The fragile X mental retardation protein (FMRP), the product of the FMR1 gene, is responsible for the fragile X syndrome (FXS). FMRP regulates miRNA expression and is involved in miRNA-mediated gene silencing. However, the question of whether FMRP is, in turn, regulated by miRNAs remains unanswered.

Mapping cis-regulatory chromatin contacts in neural cells links neuropsychiatric disorder risk variants to target genes.

Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform an integrative analysis of chromatin interactions, open chromatin regions and transcriptomes using promoter capture Hi-C, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing, respectively, in four functionally distinct neural cell types: induced pluripotent stem cell (iPSC)-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus-like neurons and primary astrocytes.

An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila.

Being relatively simple and practical, Drosophila transgenic RNAi is the technique of top priority choice to quickly study genes with pleiotropic functions. However, drawbacks have emerged over time, such as high level of false positive and negative results. To overcome these shortcomings and increase efficiency, specificity and versatility, we develop a next generation transgenic RNAi system.

The Lysine Demethylase dKDM2 Is Non-essential for Viability, but Regulates Circadian Rhythms in .

Post-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive depending on specific pathological contexts.

Phosphatidylserine Externalization Results from and Causes Neurite Degeneration in Drosophila.

Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an "eat-me" signal allowing recognition of degenerating neurites by phagocytes. Here we show that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked.

Next-generation CRISPR/Cas9 transcriptional activation in using flySAM.

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability.

Optimized strategy for in vivo Cas9-activation in .

While several large-scale resources are available for in vivo loss-of-function studies in , an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator.

Genome editing in Drosophila melanogaster: from basic genome engineering to the multipurpose CRISPR-Cas9 system.

Nowadays, genome editing tools are indispensable for studying gene function in order to increase our knowledge of biochemical processes and disease mechanisms. The extensive availability of mutagenesis and transgenesis tools make Drosophila melanogaster an excellent model organism for geneticists. Early mutagenesis tools relied on chemical or physical methods, ethyl methane sulfonate (EMS) and X-rays respectively, to randomly alter DNA at a nucleotide or chromosomal level.