High-content Imaging-based Pooled CRISPR Screens in Mammalian Cells

Yan, Xiao‐Wei; Stuurman, Nico; Ribeiro, Sylvie; Tanenbaum, Marvin E.; Horlbeck, Max A.; Liem, Christina R; Jost, Marco; Weissman, Jonathan S.; Vale, Ronald D.

doi:10.1101/2020.06.30.179648

Cited by 17 publications

(21 citation statements)

References 45 publications

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“…Pooled CRISPR screen outperforms array‐based screen by its scalability and low cost, however, was largely restricted to standard readouts, including survival, proliferation, and FACS‐sortable markers (Hanna & Doench, 2020). Most recently, combining with microscopy‐based approaches, CRISPR screen enabled the association of subcellular phenotypes with perturbation of specific gene(s) (Wheeler et al , 2020; preprint: Yan et al , 2020). In studying regulation of gene expression, Perturb‐seq, CRISP‐seq, and CROP‐seq, which combine CRISPR‐based gene editing with single‐cell mRNA sequencing, allowed transcriptome profile to serve as comprehensive molecular readout (Adamson et al , 2016; Dixit et al , 2016; Datlinger et al , 2017), but often with limited throughput.…”

Section: Introductionmentioning

confidence: 99%

CIGAR‐seq, a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

Fang

Wang

et al. 2020

Molecular Systems Biology

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Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m 6 A and m 5 C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high-throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR-seq). Using CIGAR-seq, we discovered NSUN6 as a novel mRNA m 5 C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on nonoverlapping subsets of mRNA m 5 C sites and together contributed to almost all the m 5 C modification in mRNA. Finally, using m 1 A as an example, we demonstrated that CIGAR-seq can be easily adapted for identifying regulators of other mRNA modification.

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Section: Introductionmentioning

confidence: 99%

CIGAR‐seq, a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

Fang

Wang

et al. 2020

Molecular Systems Biology

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“…We engineered two complementary Arch-derived GEVIs using a video-based, pooled screening platform ("Photopick") for mammalian cells. Pooled screens offer the practical advantage of lower cost and higher throughput compared to arrayed screens (Feldman et al, 2019;Hasle et al, 2020;Kanfer et al, 2021;Lawson et al, 2021;Lee et al, 2020;Piatkevich et al, 2018;Yan et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

Tian

Davis

Wong-Campos

et al. 2021

Preprint

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All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The “Optopatch” constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.

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“…Indeed, two recent efforts describe innovative pooled optical screen methodologies utilising cells expressing photo-convertible mCherry-based reporters. The authors described experimental pipelines of automated photoconversion of specific cells transduced with sgRNA libraries based on nucleus size [75] or protein translocation [76], followed by FACS isolation of photo-converted cells and sequencing for sgRNA enrichment. Vale et al screened~3000 genes for their effects on nucleus size, whereas Youle et al…”

Section: High-throughput Screening With Neuronal Cell Culturesmentioning

confidence: 99%

Cellular Models and High-Throughput Screening for Genetic Causality of Intellectual Disability

Fell

Nagy

2021

Trends in Molecular Medicine

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Intellectual disabilities (ID) are a type of neurodevelopmental disorder (NDD). They can have a genetic cause, including an emerging class of ID centring around Rho GTPases, such as Ras-related C3 botulinum toxin substrate 1 (RAC1). Guidelines for establishing genetic causality include the use of cellular models, which often have morphological aberrations, a long-standing hallmark of ID. Disease cellular models can facilitate high-throughput screening (HTS) of chemical or genetic perturbations, which can provide translatable biological insight. Here, we discuss a class of IDs centring around RAC1. We review novel and established cellular models of ID, including mouse and human primary cells and reprogrammed or induced neurons. Finally, we review progress and remaining challenges in the adoption of HTS methodologies by the community studying neurological disorders. Rare Intellectual Disabilities: Insights into NeurodevelopmentCommon and rare (see Glossary) forms of ID are a type of NDD that have an estimated prevalence of 2-3% in the general population, with 0.3-0.5% classified as severe ID and 85% of cases classified as mild [1,2]. Genetic studies, including next-generation sequencing (NGS) technologies, have identified 2588 genes to be involved in ID thus far [3] and the exponential rate at which this number increases each year indicates that saturation in ID gene identification has not yet been reached [2]. Indeed, a recent study estimated that >1000 genes associated with developmental disorders have yet to be identified [4]. Characterising genetic contribution to observed neuropathological features of patients is not trivial. Field-accepted guidelines for assigning genetic disease-causality require bioinformatic, statistical, and experimental approaches, including the use of cellular and animal models of disease, which are particularly challenging for neuroscientists [5]. Nevertheless, due to technical advances, common molecular pathways in seemingly distinct IDs have been identified, including an emerging class of IDs centring around RAC1. Furthermore, established and novel cellular models of common and rare neurological disease have not only contributed to our understanding of genetic neuropathology, but also paved the way for scalable genetic and pharmacological screens. HighlightsNext-generation sequencing of patients with ID continues to identify genes important to neuronal development.

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High-content Imaging-based Pooled CRISPR Screens in Mammalian Cells

Cited by 17 publications

References 45 publications

CIGAR‐seq, a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

CIGAR‐seq, a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

Cellular Models and High-Throughput Screening for Genetic Causality of Intellectual Disability

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