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 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

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. Next, we applied PRIME to functionally characterize 1,304 genome-wide association study (GWAS)-identified non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate that PRIME is capable of characterizing genetic variants at single-base resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.

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 Drosophila , these are HeT-A , TART , and TAHRE . 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 Drosophila . Depletion of multiple Mediator subunits, Dp, or Sd increased TR expression and telomere length, while over-expressing E2F1-Dp or knocking down the E2F1 regulator Rbf1 (Retinoblastoma-family protein 1) stimulated TR transcription, with Mediator and Sd affecting TR expression through E2F1-Dp. The CUT&RUN analysis revealed direct binding of CDK8, Dp, and Sd to telomeric repeats. These findings highlight the essential role of the Mediator complex in maintaining telomere homeostasis by regulating TR transcription through E2F1-Dp and Sd, revealing the intricate coupling of TR transcription with the host cell-cycle machinery, thereby ensuring chromosome end protection and genomic stability during cell division.

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. We further establish the link between functional variants and target genes by single-cell CRISPRi screening in microglia. In addition, we show that AD variants exhibit allelic imbalance on target gene expression. In particular, rs7922621 is the effective variant in controlling TSPAN14 expression among other nominated variants in the same cCRE and exerts multiple physiological effects including reduced cell surface ADAM10 and altered soluble TREM2 (sTREM2) shedding. Our work represents a systematic approach to prioritize and characterize AD-associated variants and provides a roadmap for advancing genetic association to experimentally validated cell-type-specific phenotypes and mechanisms.

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 MYC enhancer via PRIME-mediated saturation mutagenesis. Next, we applied PRIME to functionally characterize 1,304 non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate PRIME capable of characterizing genetic variants at base-pair resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.

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. The tagging strategy described in this protocol is readily applicable for knockin of other reporters in diverse cell types for biomedical research.

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. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein-RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.

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. In addition to the fact that the thermal conductivity of the vertically aligned TIMs can be further improved, another critical factor is the limited actual contact area leading to relatively high contact thermal resistance (20-30 K mm2 W-1) of the "solid-solid" mating interface formed by the vertical graphene and the rough chip/heat sink. To solve this common problem faced by vertically aligned graphene, in this work, we combined mechanical orientation and surface modification strategy to construct a three-tiered TIM composed of mainly vertically aligned graphene in the middle and micrometer-thick liquid metal as a cap layer on upper and lower surfaces. Based on rational graphene orientation regulation in the middle tier, the resultant graphene-based TIM exhibited an ultra-high thermal conductivity of 176 W m-1 K-1. Additionally, we demonstrated that the liquid metal cap layer in contact with the chip/heat sink forms a "liquid-solid" mating interface, significantly increasing the effective heat transfer area and giving a low contact thermal conductivity of 4-6 K mm2 W-1 under packaging conditions. This finding provides valuable guidance for the design of high-performance TIMs based on two-dimensional materials and improves the possibility of their practical application in electronic thermal management.

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 D, volume fraction fv, and thickness t) on the radiative properties of the common plastic materials. Through the assistance of the metal-reflective layer, the daytime cooling power Pnet of the RDSP structures is slightly higher than that of the porous structures. Without the metal-reflective layer, the porous PC films can still achieve good PDRC performance with Pnet of 86 W/m2. Furthermore, the effective thermal conductivity of different structures was evaluated. The single-layer porous structure with optimally designed architecture can achieve both good optical and insulating performance, and it is the structure with the most potential in PDRC applications. The results can provide guidelines for designing high-performance radiative cooling films.

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. However, generating null or mutant alleles is low throughput, making it impossible to characterize disease-associated variants in large quantities efficiently. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens can be leveraged to dissect the biological consequences of the tested genes and variants in their native context. Nevertheless, testing non-coding variants associated with complex diseases remains non-trivial. In this review, we first discuss the current challenges of interpreting the function of the non-coding genome and approaches to prioritizing disease-associated variants in the context of the 3D epigenome. Second, we provide a brief overview of high-throughput CRISPRi and CRISPRa screening strategies applicable for characterizing non-coding sequences in appropriate biological systems. Lastly, we discuss the promising prospects of using CRISPR-based technologies to dissect DNA sequences associated with neuropsychiatric diseases.

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-thioguanine­induced DNA mismatch repair pathway using both CRISPR interference (CRISPRi) and CRISPR nuclease (CRISPRn) approaches. Sixty percent of the identified enhancers are known promoters with distinct epigenomic features compared to other active promoters, including increased chromatin accessibility and interactivity. Furthermore, by imposing different levels of selection pressure, CRISPRpath can distinguish enhancers exerting strong impact on gene expression from those exerting weak impact. Our results offer a nuanced view of cis-regulation and demonstrate that CRISPRpath can be leveraged for understanding the complex gene regulatory program beyond transcriptional output at scale.

Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila.

The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster 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. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.

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. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFß, or Transforming Growth Factor-ß) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFß signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.

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. MAIN METHODS: We detected the FMRP expression pattern by in situ hybridization. MiR-315 overexpression and knockout models were generated by germ-line transformation and ends-out homologous recombination, respectively. Western blotting and immunohistochemistry were used to detect Drosophila FMRP (dFMRP) and a Luciferase reporter assay was used to confirm the regulation of dfmr1 mRNA by mir-315. Synaptic structural quantification and electrophysiological methods were used to compare synaptic functions among groups. KEY FINDINGS: Here, we determined that the transcription product of dFMR1, the Drosophila homologue of FMR1, is a direct target of miR-315. MiR-315 is mainly expressed in the nervous system of Drosophila. Flies overexpressing miR-315 showed pupation defects and reduced hatching rates. A homozygous miR-315 knockout status is embryonic lethal in flies. These observations indicate that miR-315 is a key regulator of the Drosophila nervous system. Furthermore, computational prediction and cell-based luciferase and in vivo assays demonstrated that dfmr1 is directly targeted by miR-315. Lastly, using the neuromuscular junction as a model, we found that miR-315 regulates synaptic structure and transmission by targeting dfmr1. SIGNIFICANCE: These findings provide compelling evidence that miR-315 targets dfmr1 in the Drosophila nervous system, acting as a regulatory factor for the fine-tuned modulation of FMRP expression.

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. We identify hundreds of thousands of long-range cis-interactions between promoters and distal promoter-interacting regions, enabling us to link regulatory elements to their target genes and reveal putative processes that are dysregulated in disease. Finally, we validate several promoter-interacting regions by using clustered regularly interspaced short palindromic repeats (CRISPR) techniques in human excitatory neurons, demonstrating that CDK5RAP3, STRAP and DRD2 are transcriptionally regulated by physically linked enhancers.

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. With this system, the leaky expression of the basal promoter is significantly reduced, as well as the heterozygous ratio of transgenic RNAi flies. In addition, it has been first achieved to precisely and efficiently modulate highly expressed genes. Furthermore, we increase versatility which can simultaneously knock down multiple genes in one step. A case illustration is provided of how this system can be used to study the synthetic developmental effect of histone acetyltransferases. Finally, we have generated a collection of transgenic RNAi lines for those genes that are highly homologous to human disease genes.

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

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. However, the role of KDM2 proteins during development remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, we have generated two dKdm2 null alleles using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately 1 h shorter than the control. Interestingly, lengthened circadian periods are observed when dKDM2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of dKDM2 and its orthologs in vertebrates regarding the regulation of circadian rhythms will advance our understanding of the epigenetic regulations of circadian clocks.

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. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. Our findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo.

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

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster 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. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.

Optimized strategy for in vivo Cas9-activation in Drosophila.

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, 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. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.