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New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Pluripotentes Induzidas , Humanos , Edição de Genes/métodos , Mutação , GenômicaRESUMO
Background: Non-motor symptoms of Parkinson's disease (PD) such as cognitive impairment are common and decrease patient quality of life and daily functioning. While no pharmacological treatments have effectively alleviated these symptoms to date, non-pharmacological approaches such as cognitive remediation therapy (CRT) and physical exercise have both been shown to improve cognitive function and quality of life in people with PD. Objective: This study aims to determine the feasibility and impact of remote CRT on cognitive function and quality of life in patients with PD participating in an organized group exercise program. Methods: Twenty-four subjects with PD recruited from Rock Steady Boxing (RSB), a non-contact group exercise program, were evaluated using standard neuropsychological and quality of life measures and randomized to the control or intervention group. The intervention group attended online CRT sessions for one hour, twice a week for 10 weeks, engaging in multi-domain cognitive exercises and group discussion. Results: Twenty-one subjects completed the study and were reevaluated. Comparing groups over time, the control group (n = 10) saw a decline in overall cognitive performance that trended towards significance (p = 0.05) and a statistically significant decrease in delayed memory (p = 0.010) and self-reported cognition (p = 0.011). Neither of these findings were seen in the intervention group (n = 11), which overwhelmingly enjoyed the CRT sessions and attested to subjective improvements in their daily lives. Conclusions: This randomized controlled pilot study suggests that remote CRT for PD patients is feasible, enjoyable, and may help slow the progression of cognitive decline. Further trials are warranted to determine the longitudinal effects of such a program.
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Autism spectrum disorder (ASD) is a heritable neurodevelopmental disorder characterized by deficits in social interactions and communication. Protein-altering variants in many genes have been shown to contribute to ASD; however, understanding the convergence across many genes remains a challenge. We demonstrate that coexpression patterns from 993 human postmortem brains are significantly correlated with the transcriptional consequences of CRISPR perturbations in human neurons. Across 71 ASD risk genes, there was significant tissue-specific convergence implicating synaptic pathways. Tissue-specific convergence was further demonstrated across schizophrenia and atrial fibrillation risk genes. The degree of ASD convergence was significantly correlated with ASD association from rare variation and differential expression in ASD brains. Positively convergent genes showed intolerance to functional mutations and had shorter coding lengths than known risk genes even after removing association with ASD. These results indicate that convergent coexpression can identify potentially novel genes that are unlikely to be discovered by sequencing studies.
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Point mutations and structural variants that directly disrupt the coding sequence of MEF2C have been associated with a spectrum of neurodevelopmental disorders (NDDs). However, the impact of MEF2C haploinsufficiency on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern its regulation. To explore the functional changes associated with structural variants that alter MEF2C expression and/or regulation, we generated an allelic series of 204 isogenic human induced pluripotent stem cell (hiPSC)-derived neural stem cells and glutamatergic induced neurons. These neuronal models harbored CRISPR-engineered mutations that involved direct deletion of MEF2C or deletion of the boundary points for topologically associating domains (TADs) and chromatin loops encompassing MEF2C. Systematic profiling of mutation-specific alterations, contrasted to unedited controls that were exposed to the same guide RNAs for each edit, revealed that deletion of MEF2C caused differential expression of genes associated with neurodevelopmental pathways and synaptic function. We also discovered significant reduction in synaptic activity measured by multielectrode arrays (MEAs) in neuronal cells. By contrast, we observed robust buffering against MEF2C regulatory disruption following deletion of a distal 5q14.3 TAD and loop boundary, whereas homozygous loss of a proximal loop boundary resulted in down-regulation of MEF2C expression and reduced electrophysiological activity on MEA that was comparable to direct gene disruption. Collectively, these studies highlight the considerable functional impact of MEF2C deletion in neuronal cells and systematically characterize the complex interactions that challenge a priori predictions of regulatory consequences from structural variants that disrupt three-dimensional genome organization.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Humanos , Genoma , Haploinsuficiência , Fatores de Transcrição MEF2/genética , Neurônios , Transcrição GênicaRESUMO
Chromosome 16p11.2 reciprocal genomic disorder, resulting from recurrent copy-number variants (CNVs), involves intellectual disability, autism spectrum disorder (ASD), and schizophrenia, but the responsible mechanisms are not known. To systemically dissect molecular effects, we performed transcriptome profiling of 350 libraries from six tissues (cortex, cerebellum, striatum, liver, brown fat, and white fat) in mouse models harboring CNVs of the syntenic 7qF3 region, as well as cellular, transcriptional, and single-cell analyses in 54 isogenic neural stem cell, induced neuron, and cerebral organoid models of CRISPR-engineered 16p11.2 CNVs. Transcriptome-wide differentially expressed genes were largely tissue-, cell-type-, and dosage-specific, although more effects were shared between deletion and duplication and across tissue than expected by chance. The broadest effects were observed in the cerebellum (2,163 differentially expressed genes), and the greatest enrichments were associated with synaptic pathways in mouse cerebellum and human induced neurons. Pathway and co-expression analyses identified energy and RNA metabolism as shared processes and enrichment for ASD-associated, loss-of-function constraint, and fragile X messenger ribonucleoprotein target gene sets. Intriguingly, reciprocal 16p11.2 dosage changes resulted in consistent decrements in neurite and electrophysiological features, and single-cell profiling of organoids showed reciprocal alterations to the proportions of excitatory and inhibitory GABAergic neurons. Changes both in neuronal ratios and in gene expression in our organoid analyses point most directly to calretinin GABAergic inhibitory neurons and the excitatory/inhibitory balance as targets of disruption that might contribute to changes in neurodevelopmental and cognitive function in 16p11.2 carriers. Collectively, our data indicate the genomic disorder involves disruption of multiple contributing biological processes and that this disruption has relative impacts that are context specific.