Detection of gene cis-regulatory element perturbations in single-cell transcriptomes

We introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq), a method that enables the direct observation of guide RNAs (gRNAs) in scRNA-seq. We use pAC-Seq to assess the phenotypic consequences of CRISPR/Cas9 based alterations of gene cis-regulatory regions. We show that pAC-Seq is able to detect cis-regulatory-induced alteration of target gene expression even when biallelic loss of target gene expression occurs in only ~5% of cells. This low rate of biallelic loss significantly increases the number of cells required to detect the consequences of changes to the regulatory genome, but can be ameliorated by transcript-targeted sequencing. Based on our experimental results we model the power to detect regulatory genome induced transcriptomic effects based on the rate of mono/biallelic loss, baseline gene expression, and the number of cells per target gRNA.

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CRISPR-Cas9 Genome Editing of a Single Regulatory Element Nearly Abolishes Target Gene Expression in Mice—Brief Report

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.114.305017 ◽

2015 ◽

Vol 35 (2) ◽

pp. 312-315 ◽

Cited By ~ 35

Author(s):

Yu Han ◽

Orazio J. Slivano ◽

Christine K. Christie ◽

Albert W. Cheng ◽

Joseph M. Miano

Keyword(s):

Gene Expression ◽

Genome Editing ◽

Target Gene ◽

Regulatory Element ◽

Target Gene Expression

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The BMP signaling gradient is interpreted through concentration thresholds in dorsal–ventral axial patterning

PLoS Biology ◽

10.1371/journal.pbio.3001059 ◽

2021 ◽

Vol 19 (1) ◽

pp. e3001059

Author(s):

Hannah Greenfeld ◽

Jerome Lin ◽

Mary C. Mullins

Keyword(s):

Gene Expression ◽

Single Cell ◽

Target Gene ◽

Target Genes ◽

Gene Activation ◽

Spatial Relationship ◽

Bmp Signaling ◽

Target Gene Expression ◽

Gradient Slope ◽

Distinct Cell

Bone Morphogenetic Protein (BMP) patterns the dorsal–ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis.

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Osthol ameliorates fat milk-induced fatty liver in mice by regulation of hepatic sterol regulatory element-binding protein-1c/2-mediated target gene expression

European Journal of Pharmacology ◽

10.1016/j.ejphar.2011.05.014 ◽

2011 ◽

Vol 666 (1-3) ◽

pp. 183-188 ◽

Cited By ~ 17

Author(s):

Rao Du ◽

Jie Xue ◽

Heng-bin Wang ◽

Yan Zhang ◽

Mei-lin Xie

Keyword(s):

Gene Expression ◽

Fatty Liver ◽

Target Gene ◽

Binding Protein ◽

Regulatory Element ◽

Target Gene Expression ◽

Element Binding Protein ◽

Sterol Regulatory Element

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2049-P: The Chd4 Helicase of the Nucleosome Remodeling and Deacetylase Complex Modulates Pdx1 Target Gene Expression In Vitro

Diabetes ◽

10.2337/db20-2049-p ◽

2020 ◽

Vol 69 (Supplement 1) ◽

pp. 2049-P

Author(s):

REBECCA K. DAVIDSON ◽

NOLAN CASEY ◽

JASON SPAETH

Keyword(s):

Gene Expression ◽

Target Gene ◽

Nucleosome Remodeling ◽

Target Gene Expression

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On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11269-z ◽

2021 ◽

Author(s):

Philipp Moritz Fricke ◽

Angelika Klemm ◽

Michael Bott ◽

Tino Polen

Keyword(s):

Gene Expression ◽

Acetic Acid ◽

Literature Search ◽

Target Gene ◽

Target Genes ◽

Recombinant Dna ◽

Acetic Acid Bacteria ◽

Homoserine Lactone ◽

Expression Systems ◽

Target Gene Expression

Abstract Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. Key points • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

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The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus

Nature Communications ◽

10.1038/s41467-020-20306-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Mohammad M. Karimi ◽

Ya Guo ◽

Xiaokai Cui ◽

Husayn A. Pallikonda ◽

Veronika Horková ◽

...

Keyword(s):

Gene Expression ◽

T Cell ◽

Single Cell ◽

Signal Strength ◽

Gene Activity ◽

Cytotoxic T Cell ◽

Individual Selection ◽

Cell Lineages ◽

Single Cell Rna Sequencing ◽

Tcr Signals

AbstractCD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.

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