Versatile phenotype-activated cell sorting

Lee, Jihwan; Suzuki, Peter; Ahrens, John F.; Lai, Syeling; Lu, Xiaoyu; Guan, Sihui; St-Pierre, François

doi:10.1126/sciadv.abb7438

Cited by 45 publications

(49 citation statements)

References 67 publications

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“…For example, the forward genetic screen described here could be readily extended to study pathological cardiomyocyte hypertrophy using the same Myh7-YFP reporter, which is reactivated by pathological cardiomyocyte stress. The scope of accessible phenotypes could be dramatically expanded by developing methods to sort individual cells by morphology 48 . Among the validated transcriptional and epigenetic regulators of CM maturation uncovered by our screen are Rnf20, Rnf40, Taf2, and Taf3.…”

Section: Discussionmentioning

confidence: 99%

Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

VanDusen

Lee

et al. 2021

Nat Commun

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The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

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Section: Discussionmentioning

confidence: 99%

Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

VanDusen

Lee

et al. 2021

Nat Commun

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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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“…In this study we utilized FACS for high-throughput screening of mammalian cells, however any other method for fluorescence cells screening can be integrated into the described workflow. For example, SPOTlight approach 52 , microfluidc sorter 48 , photostick technique 53 , or robotic cell picker 33 can be implemented following FACS enrichment of transfected cells, thus enabling phenotyping of single cells for a diversity of biochemical and photophysical properties of expressed mutated proteins, e.g. subcellular localization, pH stability, photostability, quantum yield etc.…”

Section: Discussionmentioning

confidence: 99%

Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

Babakhanova

Jung

Namikawa

et al. 2021

Preprint

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In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2x107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

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Versatile phenotype-activated cell sorting

Cited by 45 publications

References 67 publications

Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

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

Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

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