Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development

Cell, Journal Year: 2024, Volume and Issue: 187(13), P. 3236 - 3248.e21

Published: May 20, 2024

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains peripheral nervous systems. Through extensive tests 86 vectors AAV serotypes combined a transposon system, substantially amplified efficacy accelerated gene delivery from weeks days. Our proof-of-principle utero screen identified pleiotropic effects Foxg1, highlighting its tight regulation distinct networks essential for cell fate specification Layer 6 corticothalamic neurons. Notably, our platform can label >6% cerebral cells, surpassing current state-of-the-art at <0.1% by lentivirus, achieve analysis over 30,000 cells one experiment enable massively parallel Perturb-seq. Compatible various phenotypic measurements (single-cell or spatial multi-omics), it presents flexible approach interrogate function types vivo, translating variants their causal function.

Language: Английский

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Vectors in CRISPR Gene Editing for Neurological Disorders: Challenges and Opportunities

Advanced Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 14, 2025

Abstract Diseases of the nervous system are recognized as second leading cause death worldwide. The global prevalence neurological diseases, such Huntington's disease, Alzheimer's and Parkinson's disease has seen a significant rise due to increasing proportion aging population. discovery clustered regularly interspaced short palindromic repeats (CRISPR) genome editing technique paved way for universal diseases treatment. However, finding safe effective method deliver CRISPR gene‐editing tools remains main challenge therapies in vivo. Adeno‐associated virus (AAV) is currently one most commonly used vector systems, but some issues remain unresolved, including capsid immunogenicity, off‐target mutations, potential genotoxicity. To address these concerns, researchers actively encouraging development new delivery like virus‐like particles nanoparticles. These novel systems have enhance targeting efficiency, thereby offering possible solutions current challenges. This article reviews vectors disorders treatment explores overcome limitations systems. Additionally, strategies highlighted valuable studying challenges opportunities that present.

Language: Английский

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Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Feb. 20, 2024

Abstract Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors ECM clearance, relatively little is understood regarding upstream regulation this process. Collagen most abundant constituent normal and mammalian tissues. Its catabolism occurs through proteolysis cell-mediated uptake collagen fragments for intracellular degradation. Given paucity information latter process, here we execute unbiased genome-wide screens to understand molecular underpinnings clearance. Using approach, discover mechanism which biosynthesis sensed by cells internally directly regulates collagen. The sensing appears be dependent on endoplasmic reticulum-resident protein SEL1L via noncanonical function protein. This pathway functions homeostatic negative feedback loop that limits accumulation In human lung disease, induction synthesis impaired, thereby contributing pathological tissue. Thus, describe cell-autonomous, rheostatic an important tissue homeostasis.

Language: Английский

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Gene editing as a therapeutic strategy for spinocerebellar ataxia type-3

Revue Neurologique, Journal Year: 2024, Volume and Issue: 180(5), P. 378 - 382

Published: April 4, 2024

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a neurodegenerative disease caused by expanded polyglutamine repeats in exon 10 of the ataxin-3 gene, ATXN3. The accumulation mutant ATXN3 protein leads to severe clinical manifestations and premature death. Clinically, SCA3 pathology characterized progressive leading motor incoordination that may affect balance, gait speech, neuropathologically degeneration spinal cord cerebellum, well cerebral cortex basal ganglia. Although rare it most common autosomal dominant spinocerebellar worldwide. Its geographical distribution varies worldwide, with peak prevalence certain regions Brazil, Portugal China. In 1994, identification expansion gene made possible not only diagnose this but dissect mechanisms cellular degeneration. As monogenic for which symptomatic treatment available, represents an attractive therapeutic target editing strategies.

Language: Английский

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Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and Genetically Encoded Tool Delivery in the Central and Peripheral Nervous Systems

eNeuro, Journal Year: 2024, Volume and Issue: 11(7), P. ENEURO.0438 - 23.2024

Published: June 13, 2024

CRISPR/Cas9 gene editing represents an exciting avenue to study genes of unknown function and can be combined with genetically encoded tools such as fluorescent proteins, channelrhodopsins, DREADDs, various biosensors more deeply probe the these in different cell types. However, current strategies also manipulate or visualize edited cells are challenging due large size Cas9 proteins limited packaging capacity adeno-associated viruses (AAVs). To overcome constraints, we developed alternative strategy using a single AAV vector mouse lines that express Cre-dependent achieve efficient cell-type specific across nervous system. Expressing from genomic locus affords space package guide RNAs for together Cre-dependent, manipulate, map, monitor neurons virus. We validated this three common neuroscience: ChRonos, channelrhodopsin, studying synaptic transmission optogenetics, GCaMP8f recording Ca 2+ transients photometry, mCherry tracing axonal projections. tested multiple brain regions types, including GABAergic nucleus accumbens, glutamatergic projecting ventral pallidum lateral habenula, dopaminergic tegmental area, proprioceptive periphery. This flexible approach could help identify test novel affecting transmission, circuit activity, morphology viral injection.

Language: Английский

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CRISPR-based genetic screens in human pluripotent stem cells derived neurons and brain organoids

Cell and Tissue Research, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 25, 2024

Language: Английский

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Massively parallelin vivoPerturb-seq reveals cell type-specific transcriptional networks in cortical development

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Sept. 18, 2023

Abstract Systematic analysis of gene function across diverse cell types in vivo is hindered by two challenges: obtaining sufficient cells from live tissues and accurately identifying each cell’s perturbation high-throughput single-cell assays. Leveraging AAV’s versatile type tropism high labeling capacity, we expanded the resolution scale CRISPR screens: allowing phenotypic at a multitude embryonic brain, adult peripheral nervous system. We undertook extensive tests 86 AAV serotypes, combined with transposon system, to substantially amplify accelerate delivery weeks days. Using this platform, performed an utero genetic screen as proof-of-principle identified pleiotropic regulatory networks Foxg1 cortical development, including Layer 6 corticothalamic neurons where it tightly controls distinct essential for fate specification. Notably, our platform can label >6% cerebral cells, surpassing current state-of-the-art efficacy <0.1% (mediated lentivirus), achieve over 30,000 one experiment, thus enabling massively parallel Perturb-seq. Compatible various techniques (CRISPRa/i) measurements (single-cell or spatial multi-omics), presents flexible, modular approach interrogate , connecting variants their causal functions.

Language: Английский

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CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(22), P. 16161 - 16161

Published: Nov. 10, 2023

Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the progression. Induced pluripotent stem cells (iPSCs) hold potential since they sourced from adult tissue have capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on lineage patterning factors supplied in media. Genetic engineering of iPSC with introduction recombinant master regulators neuron (MN) has shorten streamline developmental programs. We established stable lines transient induction exogenous LHX3 ISL1 Tet-activator regulatory region demonstrated that transgenes is not sufficient development mature MNs absence factors. Comparative global transcriptome analysis MN native Lhx-ISL1 modified cultures genetic manipulation helped neuronal process. However, leaky gene expression resulted premature activation pathways characteristic function. Dysregulation metabolic within affected electrophysiological responses.

Language: Английский

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The long road to the development of stem cells as a model for central nervous system health and disease

Advances in neurotoxicology, Journal Year: 2024, Volume and Issue: unknown, P. 1 - 45

Published: Jan. 1, 2024

Language: Английский

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Shaping agricultural future: a comprehensive review on crispr technology applications in agriculture

Revista Brasileira de Engenharia de Biossistemas, Journal Year: 2024, Volume and Issue: 18

Published: Dec. 23, 2024

With the increasing global demand for food, new pathways have emerged to drive development and manipulation of crops with desired traits ensure food security. CRISPR technology has enabled genome editing, allowing addition or destruction specific DNA sequences modify a function, stands out as an exceptionally versatile tool. The components system include Cas9 enzyme, responsible double-strand cuts, guide RNA (gRNA), forming part spacer RNA. Through systematic review, we searched, identifying 30 articles related technique its application in agriculture. CRISPR-Cas9 been widely employed understand transcriptional regulation, make epigenetic modifications, microscopically visualize loci. results support specificity editing CRISPR/Cas9 system, demonstrating efficiency enhancing crop performance, enabling generation plants free foreign DNA, avoiding off-target mutations. Overall, increased productivity, water stress resistance, weed control various evaluated. It plays pivotal role boosting agricultural creation adapted adverse environments, significantly thus represents fundamental tool genetic engineering, propelling significant innovations address challenges.

Language: Английский

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