CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming

Liu, Yanxia; Chen, Yu; Daley, Timothy; Wang, Fangyuan; Cao, William S.; Bhate, Salil; Lin, Xueqiu; Still, Chris; Liu, Honglei; Zhao, David; Wang, Haifeng; Xie, Xinmin; Ding, Sheng; Wong, Wing Hung; Wernig, Marius; Qi, Lei

doi:10.1016/j.stem.2018.09.003

Cited by 171 publications

(142 citation statements)

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“…In addition to the Ngn2-driven generation of glutamatergic neurons (Fernandopulle et al, 2018;Wang et al, 2017;Zhang et al, 2013) used here, induced expression of different transcription factors yield other types of neurons, such as motor neurons (Hester et al, 2011;Shi et al, 2018) and inhibitory neurons (Yang et al, 2017). Systematic screens are beginning to uncover additional combinations of transcription factors driving specific neuronal fates (Liu et al, 2018;Tsunemoto et al, 2018). Thus, iPSC technology could be used to generate different neuron types from an isogenic parental cell line, which would facilitate parallel CRISPR screens to dissect neuronal subtype specific gene function.…”

Section: Discussionmentioning

confidence: 94%

CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons

Tian

Gachechiladze

Ludwig

et al. 2019

Preprint

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CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSCderived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.2 4 systematic dissection of normal and disease states of neurons, and highlight the potential of interrogating human cell biology and gene function in iPSC-derived differentiated cell types. RESULTS Robust CRISPR interference in human iPSC-derived neuronsAs a first step towards a high-throughput screening platform in neurons, we developed a scalable CRISPRi-based strategy for robust knockdown of endogenous genes in homogeneous populations of human iPSC-derived neurons. We built on our previously described i 3 Neuron (i 3 N) platform, which enables large-scale production of iPSC-derived glutamatergic neurons. Central to this platform is an iPSC line with an inducible Neurogenin 2 (Ngn2) expression cassette in the AAVS1 safe-harbor locus (Fernandopulle et al., 2018;Wang et al., 2017). To enable stable CRISPRi in iPSC-derived neurons, we generated a plasmid (pC13N-dCas9-BFP-KRAB) to insert an expression cassette for CAG promoter-driven dCas9-BFP-KRAB into the CLYBL safe harbor locus, which enables robust transgene expression throughout neuronal differentiation at higher levels than the AAVS1 locus (Cerbini et al., 2015) ( Fig. 1A). We then integrated this cassette into our i 3 N iPSC line, and called the resulting monoclonal line CRISPRi-i 3 N iPSCs. A normal karyotype was confirmed for CRISPRi-i 3 N iPSCs ( Fig. S1A).To validate CRISPRi activity, we transduced these iPSCs with a lentiviral construct expressing an sgRNA targeting the transferrin receptor gene (TFRC). Knockdown of TFRC mRNA was robust in iPSCs and in i 3 Neurons for several weeks after differentiation (Fig. 1B,C). We also validated knockdown of three additional genes, UBQLN2 (Fig. 1D,E), GRN (Fig. 1F,G) and CDH2 (Fig. S1B) by qRT-PCR, Western blot, and/or immunofluorescence. Our platform thus enables potent CRISPRi knockdown of endogenous genes in iPSC-derived neurons.Since CRISPRn-associated DNA damage has been found to be highly toxic to iPSCs (Ihry ...

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Section: Discussionmentioning

confidence: 94%

CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons

Tian

Gachechiladze

Ludwig

et al. 2019

Preprint

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“…CRISPRa is a powerful approach to elucidate the cellular and genetic basis of various biological phenomena in bacteria and higher organisms (Konermann et al, 2015;Liu et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

Fang

Cheung

et al. 2020

Preprint

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Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation have been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by up to 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.

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“…While further (meta-)analysis will likely shed more light on these different views, it is not surprising that the most efficient and reliable conversion strategies involve the combined expression of Ascl1 and Ngn2 [9,10,31,35]. Recently, it has been suggested that a huge variety of TF combinations can be applied to generate subtype-specific iNs from fibroblasts (Table 1), and TF screening studies for iN conversion have led to the identification of additional pro-neuronal factors, such as Brn3a/b/c, Brn4s, and Ezh2 [36,37]. Interestingly, differences in TF choice were noted between species, as, for example, Neu-roD1 and Ngn2 were used predominantly in human iN protocols and not in rodent protocols, but no mouse-specific or human-specific TF combinations have been established thus far [6,25].…”

Section: Enabling In Conversionmentioning

confidence: 99%

“…Further, unlike for most other human cell types, primary human neuronal cultures of defined brain regions are not available for direct comparison, which further complicates the definition of gold standard criteria. By any means, the value of cell models should be measured by how well they perform their respective tasks, and these tasks might range from teaching us more about a complex disease, or how well they integrate into a neuronal circuit following transplantation [6,[33][34][35][36][37][38]114,115].…”

Section: Updating Our Criteria To Define Neuronsmentioning

confidence: 99%

Next‐generation disease modeling with direct conversion: a new path to old neurons

2019

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Within just over a decade, human reprogramming-based disease modeling has developed from a rather outlandish idea into an essential part of disease research. While iPSCs are a valuable tool for modeling developmental and monogenetic disorders, their rejuvenated identity poses limitations for modeling age-associated diseases. Direct cell-type conversion of fibroblasts into induced neurons (iNs) circumvents rejuvenation and preserves hallmarks of cellular aging. iNs are thus advantageous for modeling diseases that possess strong age-related and epigenetic contributions and can complement iPSCbased strategies for disease modeling. In this review, we provide an overview of the state of the art of direct iN conversion and describe the key epigenetic, transcriptomic, and metabolic changes that occur in converting fibroblasts. Furthermore, we summarize new insights into this fascinating process, particularly focusing on the rapidly changing criteria used to define and characterize in vitro-born human neurons. Finally, we discuss the unique features that distinguish iNs from other reprogramming-based neuronal cell models and how iNs are relevant to disease modeling.

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CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming

Cited by 171 publications

References 50 publications

CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons

CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

Next‐generation disease modeling with direct conversion: a new path to old neurons

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