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A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

ABSTRACT: A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

PROVIDER: PRJNA503119 | ENA |

REPOSITORIES: ENA

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A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation

Project description:Empirical optimization of stem cell differentiation protocols is time consuming, is labor intensive, and typi- cally does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet sin- gle-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA ‘‘barcodelets’’ to identify them on the basis of the treatments they receive. We apply barRNA-seq to simultaneously manipulate up to seven developmental pathways and study effects on embryonic stem cell (ESC) germ layer specification and mesodermal specification, uncovering combina- torial effects of signaling pathway activation on gene expression. We further develop a data-driven frame- work for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNA-seq gastrulation atlas, and use this approach to guide ESC differen- tiation into a notochord-like population. We expect that barRNA-seq will have broad utility for investigating Q2 and understanding how cooperative signaling pathways drive cell fate acquisition.

2020-05-26 | GSE122009 | GEO

A Multiplexed Barcodelet Single-Cell RNA-Seq Approach Elucidates Combinatorial Signaling Pathways that Drive ESC Differentiation.

Project description:Empirical optimization of stem cell differentiation protocols is time consuming, is laborintensive, and typically does not comprehensively interrogate all relevant signaling pathways. Here we describe barcodelet single-cell RNA sequencing (barRNA-seq), which enables systematic exploration of cellular perturbations by tagging individual cells with RNA "barcodelets" to identify them on the basis of the treatments they receive. We apply barRNA-seq to simultaneously manipulate up to seven developmental pathways and study effects on embryonic stem cell (ESC) germ layer specification and mesodermal specification, uncovering combinatorial effects of signaling pathway activation on gene expression. We further develop a data-driven framework for identifying combinatorial signaling perturbations that drive cells toward specific fates, including several annotated in an existing scRNA-seq gastrulation atlas, and use this approach to guide ESC differentiation into a notochord-like population. We expect that barRNA-seq will have broad utility for investigating and understanding how cooperative signaling pathways drive cell fate acquisition.

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An arrayed CRISPR library for individual, combinatorial and multiplexed gene knockout III

Project description:We have combined a machine-learning approach with other strategies to optimize the efficiency of sgRNAs for CRISPR screens and have constructed a genome-wide, sequence-verified, arrayed CRISPR library. This incorporates expression strategies to facilitate multiplexed or combinatorial screening. By conducting parallel loss-of-function screens, we compare our approach to existing sgRNA design and expression strategies.

2017-07-20 | GSE97433 | GEO

An arrayed CRISPR library for individual, combinatorial and multiplexed gene knockout II

2017-07-20 | GSE97432 | GEO

An arrayed CRISPR library for individual, combinatorial and multiplexed gene knockout I

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An arrayed CRISPR library for individual, combinatorial and multiplexed gene knockout

Project description:An arrayed CRISPR library for individual, combinatorial and multiplexed gene knockout

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Multiplexed inhibition of immunosuppressive genes with Cas13d for on-demand combinatorial cancer immunotherapy

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Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems

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Systems approach elucidates convoluted transcriptional regulation on carbon metabolism

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Optimization of Cas12a for multiplexed genome- scale transcriptional activation

Project description:Cas12a CRISPR technology, unlike Cas9, allows for multiplexing guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and we provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

2023-05-08 | GSE230445 | GEO

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