Structural and transcriptional changes during early brain maturation follow fixed developmental programs defined by genetics. However, whether this is true for functional network activity remains ...unknown, primarily due to experimental inaccessibility of the initial stages of the living human brain. Here, we developed human cortical organoids that dynamically change cellular populations during maturation and exhibited consistent increases in electrical activity over the span of several months. The spontaneous network formation displayed periodic and regular oscillatory events that were dependent on glutamatergic and GABAergic signaling. The oscillatory activity transitioned to more spatiotemporally irregular patterns, and synchronous network events resembled features similar to those observed in preterm human electroencephalography. These results show that the development of structured network activity in a human neocortex model may follow stable genetic programming. Our approach provides opportunities for investigating and manipulating the role of network activity in the developing human cortex.
Display omitted
•Long-term, single-cell transcriptomics reveals cortical organoid developmental dynamics•Cortical organoids exhibit phase-amplitude coupling during network-synchronous events•Differential role of glutamate and GABA in initiating and maintaining oscillations•Network-level events are similar to the human preterm neonatal EEG features
Oscillatory activity is a candidate mechanism for how neural populations are temporally organized. Cortical organoids exhibit periodic and highly regular nested oscillatory network events that are dependent on glutamatergic and GABAergic signaling. The emerging development of network activity transitions to more spatiotemporally complex activity, capturing features of preterm infant electroencephalography.
Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington’s disease, and ...C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
Display omitted
•RNA-targeting Cas9 (RCas9) supports efficient targeting of repetitive RNAs•An RNA endonuclease fused to nuclease-null Cas9 enables an RNA-specific CRISPR system•An RCas9 system with truncated Cas9 can be packaged in adeno-associated virus•RCas9 reverses splicing defects in myotonic dystrophy type 1 patient cells
An RNA-targeting Cas9 system induces degradation of microsatellite repeat expansion RNAs, highlighting the potential of RNA-targeting CRISPR systems for therapeutic purposes.
RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To ...address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.
Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation ...in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic.
RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression, and aberrant RBP-RNA interactions can promote cancer progression. Here, we interrogate the function of ...RBPs in cancer using pooled CRISPR-Cas9 screening and identify 57 RBP candidates with distinct roles in supporting MYC-driven oncogenic pathways. We find that disrupting YTHDF2-dependent mRNA degradation triggers apoptosis in triple-negative breast cancer (TNBC) cells and tumors. eCLIP and m6A sequencing reveal that YTHDF2 interacts with mRNAs encoding proteins in the MAPK pathway that, when stabilized, induce epithelial-to-mesenchymal transition and increase global translation rates. scRibo-STAMP profiling of translating mRNAs reveals unique alterations in the translatome of single cells within YTHDF2-depleted solid tumors, which selectively contribute to endoplasmic reticulum stress-induced apoptosis in TNBC cells. Thus, our work highlights the therapeutic potential of RBPs by uncovering a critical role for YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven breast cancers.
Display omitted
•RBP CRISPR screening identifies 57 candidates that maintain MYC-driven cell survival•YTHDF2 depletion triggers apoptosis in triple-negative breast cancer cells and tumors•YTHDF2 maintains mRNA homeostasis by limiting the number of translating mRNAs•scRibo-STAMP reveals proteomic heterogeneity in triple-negative breast tumors in vivo
RNA-binding proteins (RBPs) are aberrantly expressed in cancer, but they remain largely unexplored as drug targets. Using RBP-focused CRISPR-Cas9 screening, Einstein et al. uncover an essential and selective role for YTHDF2 in MYC-driven breast cancer, highlighting the utility of discovering RBPs as safe and effective therapeutic targets.
The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative ...disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1) can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyladenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H₂O₂, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.
The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for ...measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.
Although DNA repair is known to impact susceptibility to cancer and other diseases, relatively few population studies have been performed to evaluate DNA repair kinetics in people due to the ...difficulty of assessing DNA repair in a high-throughput manner. Here we use the CometChip, a high-throughput comet assay, to explore inter-individual variation in repair of oxidative damage to DNA, a known risk factor for aging, cancer and other diseases. DNA repair capacity after H2O2-induced DNA oxidation damage was quantified in peripheral blood mononuclear cells (PBMCs). For 10 individuals, blood was drawn at several times over the course of 4–6 weeks. In addition, blood was drawn once from each of 56 individuals. DNA damage levels were quantified prior to exposure to H2O2 and at 0, 15, 30, 60, and 120-min post exposure. We found that there is significant variability in DNA repair efficiency among individuals. When subdivided into quartiles by DNA repair efficiency, we found that the average t1/2 is 81 min for the slowest group and 24 min for the fastest group. This work shows that the CometChip can be used to uncover significant differences in repair kinetics among people, pointing to its utility in future epidemiological and clinical studies.
Display omitted
•The comet assay can be used to measure DNA oxidation damage.•CometChip is a higher throughput DNA damage assay.•CometChip can be used to measure repair kinetics.•Repair rates have been measured in dividing lymphocytes (PBMCs).•Repair capacity varies for PBMCs from different individuals.
The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (
) plays a key role in neural development and function.
also includes a protein-coding difference between the ...modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of
Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in
, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.
PDF
