Identification and characterisation of novel targets for treatment is a priority in the field of psychiatry.FKBP5is a gene with decades of evidence suggesting its pathogenic role in a subset of psychiatric patients, with potential to be leveraged as a therapeutic target for these individuals. While it is widely reported thatFKBP5/FKBP51 mRNA/protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, it is not known in which cell types and sub-anatomical areas of the human brain this occurs. This knowledge is critical to propelFKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n = 1024) ofFKBP5/1, examining neocortical areas (BA9, BA11 and ventral BA24/BA24a) derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease state, ageing and genotype on corticalFKBP5/1 expression including in a cell type-specific manner. We identified consistently heightenedFKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq; BA9 and BA11) and targeted histology (BA9, BA24a), we established that these disease and ageing effects onFKBP5/1 expression were most pronounced in excitatory superficial layer neurons of the neocortex, and this effect appeared to be consistent in both the granular and agranular areas examined. We then found that this increase inFKBP5levels may impact on synaptic plasticity, asFKBP5gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in superficial layer neurons in BA11. These findings pinpoint a novel cellular and molecular mechanism that has potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.

The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs. The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.

Motivation: Diseases and adverse drug reactions are frequently caused by disruptions in gene functionality. Gaining insight into the global system properties governing the relationships between genotype and phenotype is thus crucial to understand and interfere with perturbations in complex organisms such as diseases states. Results: We present a systematic analysis of phenotypic information of 5047 perturbations of single genes in mice, 4766 human diseases and 1666 drugs that examines the relationships between different gene properties and the phenotypic impact at the organ system level in mammalian organisms. We observe that while single gene perturbations and alterations of nonessential, tissue-specific genes or those with low betweenness centrality in protein–protein interaction networks often show organ-specific effects, multiple gene alterations resulting e.g. from complex disorders and drug treatments have a more widespread impact. Interestingly, certain cellular localizations are distinctly associated to systemic effects in monogenic disease genes and mouse gene perturbations, such as the lumen of intracellular organelles and transcription factor complexes, respectively. In summary, we show that the broadness of the phenotypic effect is clearly related to certain gene properties and is an indicator of the severity of perturbations. This work contributes to the understanding of gene properties influencing the systemic effects of diseases and drugs. Contact:monica.campillos@helmholtz-muenchen.de Supplementary information: Supplementary data are available at Bioinformatics online.

Despite recent advances in the treatment of non-small cell lung cancer (NSCLC), acquired drug resistance to targeted therapy remains a major obstacle. Epithelial-mesenchymal transition (EMT) has been identified as a key resistance mechanism in NSCLC. Here, we investigated the mechanistic role of key EMT-regulating small non-coding microRNAs (miRNAs) in sublines of the NSCLC cell line HCC4006 adapted to afatinib, erlotinib, gefitinib, or osimertinib. The most differentially expressed miRNAs derived from extracellular vesicles were associated with EMT, and their predicted target ZEB1 was significantly overexpressed in all resistant cell lines. Transfection of a miR-205-5p mimic partially reversed EMT by inhibiting ZEB1, restoring CDH1 expression, and inhibiting migration in erlotinib-resistant cells. Gene expression of EMT-markers, transcription factors, and miRNAs were correlated during stepwise osimertinib adaptation of HCC4006 cells. Temporally relieving cells of osimertinib reversed transition trends, suggesting that the implementation of treatment pauses could provide prolonged benefits for patients. Our results provide new insights into the contribution of miRNAs to drug-resistant NSCLC harboring EGFR-activating mutations and highlight their role as potential biomarkers and therapeutic targets.

Bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia share genetic architecture, yet their molecular mechanisms remain elusive. Both common and rare genetic variants contribute to neural dysfunction, impacting cognition and behavior. This study investigates the molecular effects of genetic variants on human cortical single-cell types using a single-exon analysis approach. Integrating exon-level eQTLs (common variants influencing exon expression) and joint exon eQT-Scores (combining polygenic risk scores with exon-level gene expression) from a postmortem psychiatric cohort (BD = 15, MDD = 24, schizophrenia = 68, controls = 62) with schizophrenia-focused rare variant data from the SCHEMA consortium, we identified 110 core genes enriched in pathways including circadian entrainment (FDR = 0.02), cortisol synthesis and secretion (FDR = 0.026), and dopaminergic synapse (FDR = 0.038). Additional enriched pathways included hormone signaling (FDRs < 0.0298, including insulin, GnRH, aldosterone, and growth hormone pathways) and, notably, adrenergic signaling in cardiomyocytes (FDR = 0.0028). These pathways highlight shared molecular mechanisms in the three disorders. Single-nuclei RNA sequencing data from three cortical regions revealed that these core set genes are predominantly expressed in excitatory neuron layers 2–6 of the dorsolateral prefrontal cortex, linking molecular changes to cell types involved in cognitive dysfunction. Our results demonstrate the power of integrating multimodal genetic and transcriptomic data at the exon level. This approach moves beyond symptom-based diagnoses toward molecular classifications, identifying potential therapeutic targets for psychiatric disorders.

Liquid biopsies are revolutionizing the detection and management of malignant diseases. While repetitive DNA sequences, such as LINE-1 and ALU are established in cell-free DNA (cfDNA) research, their clinical applications remain limited. In this study, we explore human satellite 2 (HSATII), a prevalent repeat DNA sequence in plasma that exhibits increased levels in cancer patients, thereby positioning it as a potential pan-cancer biomarker. We employed targeted sequencing and copy number variation (CNV) analysis using two primer pairs to assess the differential abundance of HSATII sequences in the plasma of breast cancer patients compared to healthy individuals. PCR amplicons of HSATII from 10 patients and 10 control subjects were sequenced, generating 151 bp paired-end reads. By constructing a pooled reference dataset, HSATII copy ratios were estimated in the patients. Our analysis revealed several significant CNVs in HSATII, with certain sequences displaying notable gains and losses across all breast cancer patients, suggesting their potential as biomarkers. However, we observed pronounced fragmentation of cfDNA in cancer, leading to the loss of longer PCR amplicons (>180 bp). While not all observed losses can be attributed to fragmentation artifacts, this phenomenon does introduce complexity in interpreting CNV data. Notably, this research marks the first instance of targeted HSATII sequencing in a liquid biopsy context. Our findings lay the groundwork for developing sequencing-based assays to detect differentially represented HSATII sequences, potentially advancing the field of minimally-invasive cancer screening.