Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.

Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms.

CRISPRi-based screen of Autism Spectrum Disorder risk genes in microglia uncovers roles of in microglia endocytosis and synaptic pruning.

Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells.

pyPAGE: A framework for Addressing biases in gene-set enrichment analysis-A case study on Alzheimer's disease.

Inferring the driving regulatory programs from comparative analysis of gene expression data is a cornerstone of systems biology. Many computational frameworks were developed to address this problem, including our iPAGE (information-theoretic Pathway Analysis of Gene Expression) toolset that uses information theory to detect non-random patterns of expression associated with given pathways or regulons. Our recent observations, however, indicate that existing approaches are susceptible to the technical biases that are inherent to most real world annotations.

An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation.

Trafficking of G protein-coupled receptors (GPCRs) through the endosomal-lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and the large number of cellular proteins involved in the process. Here, we developed a high-sensitivity biosensor for GPCR expression and agonist-induced trafficking to the lysosome by leveraging the ability of the engineered peroxidase APEX2 to activate the fluorogenic substrate Amplex UltraRed (AUR).

mTOR activation induces endolysosomal remodeling and nonclassical secretion of IL-32 via exosomes in inflammatory reactive astrocytes.

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos.

The Neurolipid Atlas: a lipidomics resource for neurodegenerative diseases uncovers cholesterol as a regulator of astrocyte reactivity impaired by ApoE4.

Lipid changes in the brain have been implicated in many neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's disease and Amyotrophic Lateral Sclerosis. To facilitate comparative lipidomic research across brain-diseases we established a data commons named the Neurolipid Atlas, that we have pre-populated with novel human, mouse and isogenic induced pluripotent stem cell (iPSC)-derived lipidomics data for different brain diseases. We show that iPSC-derived neurons, microglia and astrocytes display distinct lipid profiles that recapitulate lipotypes.

Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes.

Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity.

Molecular and cellular mechanisms of selective vulnerability in neurodegenerative diseases.

The selective vulnerability of specific neuronal subtypes is a hallmark of neurodegenerative diseases. In this Review, I summarize our current understanding of the brain regions and cell types that are selectively vulnerable in different neurodegenerative diseases and describe the proposed underlying cell-autonomous and non-cell-autonomous mechanisms. I highlight how recent methodological innovations - including single-cell transcriptomics, CRISPR-based screens and human cell-based models of disease - are enabling new breakthroughs in our understanding of selective vulnerability.

Autism genes converge on microtubule biology and RNA-binding proteins during excitatory neurogenesis.

Recent studies have identified over one hundred high-confidence (hc) autism spectrum disorder (ASD) genes. Systems biological and functional analyses on smaller subsets of these genes have consistently implicated excitatory neurogenesis. However, the extent to which the broader set of hcASD genes are involved in this process has not been explored systematically nor have the biological pathways underlying this convergence been identified.

Toward a CRISPR understanding of gene function in human brain development.

Genome-wide association studies pinpoint genetic risk factors for neurodevelopmental disorders (NDDs), but the next challenge is to understand the mechanisms through which these genes affect brain development. Two recent CRISPR screens in human brain organoids interrogate the function of risk genes for autism spectrum disorder and other NDDs.

Pharmacologic activation of a compensatory integrated stress response kinase promotes mitochondrial remodeling in PERK-deficient cells.

The integrated stress response (ISR) comprises the eIF2α kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2α kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells.

Human iPSC 4R tauopathy model uncovers modifiers of tau propagation.

Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to lack of appropriate human models. Current human induced pluripotent stem cell (hiPSC)-derived neurons express very low levels of 4-repeat (4R)-tau isoforms that are normally expressed in adult brain. Here, we engineered new iPSC lines to express 4R-tau and 4R-tau carrying the P301S mutation when differentiated into neurons.

Neurodegeneration enters the era of functional genomics.

There are no cures for the most common neurodegenerative diseases. None of the currently approved treatments cure or halt these conditions; rather, they address symptoms or slow disease progression. A focus on protein deposits in the brain-a hallmark of Alzheimer's disease (AD) and Parkinson's disease (PD)-has led to the development of immunotherapy drugs.

DELE1 oligomerization promotes integrated stress response activation.

Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer.

Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.

Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function.

CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly scalable cell type-specific screening platform.

There is a significant need for scalable CRISPR-based genetic screening methods that can be applied directly in mammalian tissues while enabling cell type-specific analysis. To address this, we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in the mouse tissues. We demonstrate the utility of this approach by screening two distinct large sgRNA libraries, together targeting over 5,000 genes, in mouse brains to create a robust profile of neuron-essential genes.

CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.

Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation and subsequent dysfunction and death, but the underlying mechanisms are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons.

Viral subversion of selective autophagy is critical for biogenesis of virus replication organelles.

Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study.

CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions.

The sheer complexity of the brain has complicated our ability to understand the cellular and molecular mechanisms underlying its function in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease.