CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons

Tian, Ruilin; Gachechiladze, Mariam A.; Ludwig, Connor; Laurie, Matthew T.; Hong, Jin-Hyuk; Nathaniel, Diane; Prabhu, Anika V.; Fernandopulle, Michael S.; Patel, Rajan; Abshari, Mehrnoosh; Ward, Michael E.; Kampmann, Martin

doi:10.1016/j.neuron.2019.07.014

Cited by 318 publications

(407 citation statements)

References 70 publications

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“…To uncover potential cell biological mechanisms underlying the vulnerability of EC RORB+ excitatory neurons, we compared the gene expression profiles of EC RORB-expressing excitatory neurons against all other EC excitatory neurons, which revealed differences in the expression of genes encoding synapse-vs. axon-localized proteins, potassium channel subunits, G-protein signaling molecules, and neurotransmitter receptor signaling molecules. Future studies utilizing in vitro and animal models of AD together with techniques for manipulating gene expression such as CRISPR inhibition and activation [61][62][63] will make it possible to address these potential mechanistic connections among RORB-expression, phospho-tau accumulation, and vulnerability. In neocortical areas, layers III and V are the first to accumulate tau neurofibrillary inclusions in AD 3,32,33 .…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

Leng

Eser

et al. 2020

Preprint

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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 individuals spanning the neuropathological progression of AD. 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. MAIN TEXTSelective vulnerability is a fundamental feature of neurodegenerative diseases, in which different neuronal populations show a gradient of susceptibility to degeneration 1, 2 . Selective vulnerability at the network level has been extensively explored in Alzheimer's disease (AD) 3-5 , currently the leading cause of dementia and lacking in effective therapies. However, little is known about the mechanisms underlying selective vulnerability at the cellular level in AD, which could provide insight into disease mechanisms and lead to therapeutic strategies.The entorhinal cortex (EC), an allocortex, is one of the first cortical brain regions to exhibit neuronal loss in AD 6 . Neurons in the external EC layers, especially in layer II (also known as alpha clusters of the lamina principalis externa, abbreviated "Pre-alpha") 7 , accumulate taupositive neurofibrillary changes and die early on in the course of AD 8-13 . However, these selectively vulnerable neurons have yet to be characterized extensively at the molecular level. Furthermore, it is unknown whether there are differences in vulnerability among subpopulations of these neurons. Although rodent models of AD have offered some insights [14][15][16] , the human brain has unique features with regard to cellular physiology, composition and aging [17][18][19] , limiting the extrapoloation of findings from animal models to address selective vulnerability.Previous studies have combined laser capture microdissection with microarray analysis of gene expression 20, 21 to characterize EC neurons in AD, but focused on disease-related changes in gene expression, rather than selective vulnerability. More recently, single-nucleus RNA-sequencing (snRNA-seq) has enabled large-scale characterization of transcriptomic profiles of individual cells from post-mortem human brain tissue 22, 23 . However, snRNA-seq studies of AD p...

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Section: Discussionmentioning

confidence: 99%

Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

Leng

Eser

et al. 2020

Preprint

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“…Although CRISPR-based screens are heavily used in oncology research (Hart et al, 2015;Tzelepis et al, 2016;Chow et al, 2017), these tools have garnered significantly less attention for large-scale genetic studies in diseaserelevant cell types such as differentiated neurons. Tian and coworkers recently performed several CRISPRi screens to elucidate functional contributions of various genes to cell survival, differentiation, transcriptional regulation and morphology in human iPSC (hiPSC)-derived neurons (Tian et al, 2019). In an initial survival screen, dCas9-BFP-KRAB and the LV H1 sgRNA library were used to target ;2300 genes comprising the "druggable genome."…”

Section: Crispr Screensmentioning

confidence: 99%

Genetically Engineering the Nervous System with CRISPR-Cas

Sandoval

Elahi

Ploski

2020

eNeuro

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The multitude of neuronal subtypes and extensive interconnectivity of the mammalian brain presents a substantial challenge to those seeking to decipher its functions. While the molecular mechanisms of several neuronal functions remain poorly characterized, advances in next-generation sequencing (NGS) and gene-editing technology have begun to close this gap. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system has emerged as a powerful genetic tool capable of manipulating the genome of essentially any organism and cell type. This technology has advanced our understanding of complex neurologic diseases by enabling the rapid generation of novel, disease-relevant in vitro and transgenic animal models. In this review, we discuss recent developments in the rapidly accelerating field of CRISPR-mediated genome engineering. We begin with an overview of the canonical function of the CRISPR platform, followed by a functional review of its many adaptations, with an emphasis on its applications for

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“…To further query the subset of potential genes contributing to AD at a high throughput level, arrayed CRISPR screens may be applied that take advantage of multi-well plates and validate the functional role each gene may have in the selected phenotype, which is further illustrated in Figure 5B (71). Although this technology is yet to be applied in AD, a proof-ofconcept experiment harnessed array screening to enable longitudinal study of CRISPRi effects in iPSC derived neurons, thereby paving the way for future applications in neurodegeneration (76). In conclusion, investigating the causality of epigenetic variation in sporadic AD is a daunting task that is hindered by the complex regulatory interactions that affect gene expression.…”

Section: Solving the Epigenetic Puzzle Of Ad: In Search Of The Causalmentioning

confidence: 99%

Applying gene‐editing technology to elucidate the functional consequence of genetic and epigenetic variation in Alzheimer’s disease

2020

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Recent studies have highlighted a potential role of genetic and epigenetic variation in the development of Alzheimer’s disease. Application of the CRISPR‐Cas genome‐editing platform has enabled investigation of the functional impact that Alzheimer’s disease‐associated gene mutations have on gene expression. Moreover, recent advances in the technology have led to the generation of CRISPR‐Cas–based tools that allow for high‐throughput interrogation of different risk variants to elucidate the interplay between genomic regulatory features, epigenetic modifications, and chromatin structure. In this review, we examine the various iterations of the CRISPR‐Cas system and their potential application for exploring the complex interactions and disruptions in gene regulatory circuits that contribute to Alzheimer’s disease.

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CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons

Cited by 318 publications

References 70 publications

Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

Molecular characterization of selectively vulnerable neurons in Alzheimer’s Disease

Genetically Engineering the Nervous System with CRISPR-Cas

Applying gene‐editing technology to elucidate the functional consequence of genetic and epigenetic variation in Alzheimer’s disease

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