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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. An understanding of the molecular mechanisms that determine selective vulnerability and resilience would shed light on the key processes that drive neurodegeneration and point to potential therapeutic strategies to protect vulnerable cell populations.Selective vulnerability of particular neuronal cell types is a characteristic of neurodegenerative diseases. Martin Kampmann explores our current understanding of the cellular and molecular mechanisms that lead to selective vulnerability in different diseases.

Alzheimer’s disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus—brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively—from postmortem brains spanning the progression of AD-type tau neurofibrillary pathology. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience. Leng et al. uncover the molecular signature of neuronal subpopulations that are selectively vulnerable to tau aggregation and death early in Alzheimer’s disease in the human entorhinal cortex and other brain regions, validating RORB as a marker.

Background The rapid adoption of CRISPR technology has enabled biomedical researchers to conduct CRISPR-based genetic screens in a pooled format. The quality of results from such screens is heavily dependent on the selection of optimal screen design parameters, which also affects cost and scalability. However, the cost and effort of implementing pooled screens prohibits experimental testing of a large number of parameters. Results We present CRISPulator, a Monte Carlo method-based computational tool that simulates the impact of screen parameters on the robustness of screen results, thereby enabling users to build intuition and insights that will inform their experimental strategy. CRISPulator enables the simulation of screens relying on either CRISPR interference (CRISPRi) or CRISPR nuclease (CRISPRn). Pooled screens based on cell growth/survival, as well as fluorescence-activated cell sorting according to fluorescent reporter phenotypes are supported. CRISPulator is freely available online ( http://crispulator.ucsf.edu ). Conclusions CRISPulator facilitates the design of pooled genetic screens by enabling the exploration of a large space of experimental parameters in silico, rather than through costly experimental trial and error. We illustrate its power by deriving non-obvious rules for optimal screen design.

Our understanding of neurological diseases has been tremendously enhanced over the past decade by the application of new technologies. Genome-wide association studies have highlighted glial cells as important players in diseases. Single-cell profiling technologies are providing descriptions of disease states of neurons and glia at unprecedented molecular resolution. However, significant gaps remain in our understanding of the mechanisms driving disease-associated cell states, and how these states contribute to disease. These gaps in our understanding can be bridged by CRISPR-based functional genomics, a powerful approach to systematically interrogate gene function. In this review, we will briefly review the current literature on neurological disease-associated cell states and introduce CRISPR-based functional genomics. We discuss how advances in CRISPR-based screens, especially when implemented in the relevant brain cell types or cellular environments, have paved the way towards uncovering mechanisms underlying neurological disease-associated cell states. Finally, we will delineate current challenges and future directions for CRISPR-based functional genomics to further our understanding of neurological diseases and potential therapeutic strategies.

Neurodegenerative, neurodevelopmental and neuropsychiatric disorders are among the greatest public health challenges, as many lack disease-modifying treatments. A major reason for the absence of effective therapies is our limited understanding of the causative molecular and cellular mechanisms. Genome-wide association studies are providing a growing catalogue of disease-associated genetic variants, and the next challenge is to elucidate how these variants cause disease and to translate this understanding into therapies. This Review describes how new CRISPR-based functional genomics approaches can uncover disease mechanisms and therapeutic targets in neurological diseases. The bacterial CRISPR system can be used in experimental disease models to edit genomes and to control gene expression levels through CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa). These genetic perturbations can be implemented in massively parallel genetic screens to evaluate the functional consequences for human cells. CRISPR screens are particularly powerful in combination with induced pluripotent stem cell technology, which enables the derivation of differentiated cell types, such as neurons and glia, and brain organoids from cells obtained from patients. Modelling of disease-associated changes in gene expression via CRISPRi and CRISPRa can pinpoint causal changes. In addition, genetic modifier screens can be used to elucidate disease mechanisms and causal determinants of cell type-selective vulnerability and to identify therapeutic targets.The search for effective therapies for neurological disease has been impeded by our limited understanding of the causative molecular and cellular mechanisms. Kampmann describes how new CRISPR-based functional genomics approaches can uncover disease mechanisms and therapeutic targets in neurological diseases.

Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the ‘druggable genome’. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting. Dräger et al. establish a rapid, scalable platform for iPSC-derived microglia. CRISPRi/a screens uncover roles of disease-associated genes in phagocytosis, and regulators of disease-relevant microglial states that can be targeted pharmacologically.

We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016 ). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites.

Combining gene activation and knockout of different genes in the same cell using two different Cas9 enzymes enables the reconstruction of directional dependency. Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair. Based on the results from over 100,000 perturbed gene pairs, we reconstruct a directional dependency network for human K562 leukemia cells and demonstrate how our approach allows the determination of directionality in activating genetic interactions. Our interaction network connects previously uncharacterized genes to well-studied pathways and identifies targets relevant for therapeutic intervention.

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function. Pathological transformation of astrocytes to reactive states can be protective or harmful to BBB function. Here, using a human induced pluripotent stem cell (iPSC)-derived BBB co-culture model, we show that tumor necrosis factor (TNF) transitions astrocytes to an inflammatory reactive state that causes BBB dysfunction through activation of STAT3 and increased expression of SERPINA3 , which encodes alpha 1-antichymotrypsin (α1ACT). To contextualize these findings, we correlated astrocytic STAT3 activation to vascular inflammation in postmortem human tissue. Further, in murine brain organotypic cultures, astrocyte-specific silencing of Serpina3n reduced vascular inflammation after TNF challenge. Last, treatment with recombinant Serpina3n in both ex vivo explant cultures and in vivo was sufficient to induce BBB dysfunction-related molecular changes. Overall, our results define the TNF-STAT3-α1ACT signaling axis as a driver of an inflammatory reactive astrocyte signature that contributes to BBB dysfunction. Inflammation of brain endothelial cells is seen in neurodegenerative conditions and in aging. Here the authors examine the role of astrocytes in blood brain barrier function using an iPSC-derived cell co-culture model.

Astrocytes become reactive in response to insults to the central nervous system by adopting context-specific cellular signatures and outputs, but a systematic understanding of the underlying molecular mechanisms is lacking. In this study, we developed CRISPR interference screening in human induced pluripotent stem cell-derived astrocytes coupled to single-cell transcriptomics to systematically interrogate cytokine-induced inflammatory astrocyte reactivity. We found that autocrine–paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures, one promoted by STAT3 and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in human brains in Alzheimer’s disease and hypoxic-ischemic encephalopathy. Furthermore, we validated that markers of these signatures were regulated by STAT3 in vivo using a mouse model of neuroinflammation. These results and the platform that we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity. Leng et al. establish CRISPRi screens in astrocytes to dissect pathways controlling inflammatory reactivity. They uncover two distinct inflammatory reactive signatures that are inversely regulated by STAT3 and validate that these exist in human disease.