CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states

Granulin loss of function in human mature brain organoids implicates astrocytes in TDP-43 pathology

“…Thus, our ability to generate mixed GRN +/+ neurons + GRN −/− astrocytes showing nearly as strong crSTMN2 and TDP-43 phenotypes of full GRN −/− neuron and astrocyte cultures indicate that GRN LoF in human astrocytes can lead to TDP-43 LoF in neurons. This finding is mechanistically important and adds to a growing body of literature suggesting that disease-associated astrocytes and more generally glia can drive cell death and neuronal dysfunction ( Huang et al., 2022 ; Leng et al, 2022 ; Liddelow et al., 2017 ; Taha et al., 2022 ; Zhang et al., 2020 ).…”

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

“…Like other CRISPR approaches, CRISPRi has been paired with large-scale sgRNA libraries to conduct systematic genetic screens. Such screens have been deployed to identify essential protein-coding and non-coding genes ( Gilbert et al, 2014 ; Haswell et al, 2021 ; Horlbeck et al, 2016a ; Liu et al, 2017 ; Raffeiner et al, 2020 ), to map the targets of regulatory elements ( Fulco et al, 2019 ; Fulco et al, 2016 ; Gasperini et al, 2019 ; Kearns et al, 2015 ; Klann et al, 2017 ; Thakore et al, 2015 ), to identify regulators of cellular signaling and metabolism ( Coukos et al, 2021 ; Liang et al, 2020 ; Luteijn et al, 2019 ; Semesta et al, 2020 ), to uncover stress response pathways in stem cell-derived neurons ( Tian et al, 2021 ; Tian et al, 2019 ), to uncover regulators of disease-associated states in microglia and astrocytes ( Dräger et al, 2022 ; Leng et al, 2022 ), to decode regulators of cytokine production in primary human T-cells ( Schmidt et al, 2022 ), to define mechanisms of action of bioactive small molecules ( Jost et al, 2017 ; Morgens et al, 2019 ; le Sage et al, 2017 ), to identify synthetic-lethal genetic interactions in cancer cells ( Du et al, 2017 ; Horlbeck et al, 2018 ), and to identify genetic determinants of complex transcriptional responses using RNA-seq readouts (Perturb-seq) ( Adamson et al, 2016 ; Replogle et al, 2022 ; Replogle et al, 2020 ; Tian et al, 2021 ; Tian et al, 2019 ), among others.…”

deCLUTTER2+pipeline to analyze calcium traces in a novel stem cell model for ventral midbrain patterned astrocytes

“…Reactive astrocytes play an important role in the pathogenesis of many neurodegenerative diseases (Escartin et al, 2021). To investigate this, several studies have modeled inflammation-stimulated reactivity in iPSC-derived astrocytes (Barbar et al, 2020; Leng et al, 2022; Perriot et al, 2018; Roybon et al, 2013; Santos et al, 2017; Tchieu et al, 2019; Tcw et al, 2017). We also characterized the transcriptomic profile of our astrocytes stimulated with pro-inflammatory cytokines TNF-α, IL-1α, and C1q that drive an A1-like reactive state (Liddelow et al, 2017) using qPCR.…”

“…Therefore, we investigated the responses of astrocytes to pro-inflammatory stimuli by treating the cells with TNF-α, IL-1α, and C1q that drive a reactive phenotype (Escartin et al, 2021; Liddelow et al, 2017). This assay has been widely used to study neuroinflammation in vitro and in vivo (Barbar et al, 2020; Leng et al, 2022; Liddelow et al, 2017; Zhou et al, 2019). Moreover, the morphology and transcriptomic profile of stimulated astrocytes have been well characterized.…”