scholarly journals Single-cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Heon Seok Kim ◽  
Susan M. Grimes ◽  
Anna C. Hooker ◽  
Billy T. Lau ◽  
Hanlee P. Ji
Keyword(s):  
Single Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Splicing Factors ◽  
Mrna Isoforms ◽  
Structure Detection ◽  
Long Read ◽  
Cell Gene Expression ◽  
Transcript Structure

AbstractWe developed a single-cell approach to detect CRISPR-modified mRNA transcript structures. This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative mRNA isoforms. We determine how alternative splicing is regulated by editing target exon-intron segments or splicing factors by CRISPR-Cas9 and their consequences on transcriptome profile. Our method combines long-read sequencing to characterize the transcript structure and short-read sequencing to match the single-cell gene expression profiles and gRNA sequence and therefore provides targeted genomic edits and transcript isoform structure detection at single-cell resolution.

scholarly journals Single cell characterization of CRISPR-modified transcript isoforms with nanopore sequencing

2021 ◽  
Author(s):  
Heon Seok Kim ◽  
Susan M Grimes ◽  
Anna C Hooker ◽  
Billy T Lau ◽  
Hanlee P Ji
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Splicing Factors ◽  
Transcript Isoforms ◽  
Long Read ◽  
Cell Gene Expression ◽  
Transcript Structure ◽  
Alternative Splicing Events

Transcript isoforms are mRNAs that arise from alternative splicing events. During RNA processing, different combinations of a gene's exons lead to a diverse set of isoforms. Polymorphisms or mutations at splice junctions can generate alternative splicing events. Various splicing factors also impact the representation of a gene's transcript isoforms. To assess how these two features contribute to alternative splicing, we developed a single cell approach to introduce CRISPR edits that modify mRNA transcript structure. Our method combines (1) long-read sequencing to characterize the expressed transcripts and identify the edit at single cell resolution; (2) short-read sequencing to match the single cell gene expression profiles of the cells with the altered isoform. First, we modify target exon-intron segments with CRISPR-Cas9. Second, using cDNAs with cell barcodes, we use long read sequencing to directly identify the changes in transcript isoforms from the targeted CRISPR edits. As a variation on this approach, we also determined how modifying specific splicing factors influence isoform expression and structure. Overall, we demonstrate how the integration of single cell long read analysis and CRISPR engineering can be used to directly confirm transcript isoform and target genomic edits at single cell resolution. This approach will improve our understanding of the role of alternative splicing in transcriptional regulation.

scholarly journals Circulating tumor cell characterization of lung cancer brain metastasis in the cerebrospinal fluid through single-cell transcriptome analysis

2020 ◽  
Author(s):  
Haoyu Ruan ◽  
Yihang Zhou ◽  
Jie Shen ◽  
Yue Zhai ◽  
Ying Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cerebrospinal Fluid ◽  
Single Cell ◽  
Tumor Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Unknown Primary ◽  
Great Promise ◽  
Cell Cycle Gene

AbstractMetastatic lung cancer accounts for about half of the brain metastases (BM). Development of leptomeningeal metastases (LM) are becoming increasingly common, and its prognosis is still poor despite the advances in systemic and local approaches. Cytology analysis in the cerebrospinal fluid (CSF) remains the diagnostic gold standard. Although several previous studies performed in CSF have offered great promise for the diagnostics and therapeutics of LM, a comprehensive characterization of circulating tumor cells (CTCs) in CSF is still lacking. To fill this critical gap of lung adenocarcinoma LM (LUAD-LM), we analyzed the transcriptomes of 1,375 cells from 5 LUAD-LM patient and 3 control samples using single-cell RNA sequencing technology. We defined CSF-CTCs based on abundant expression of epithelial markers and genes with lung origin, as well as the enrichment of metabolic pathway and cell adhesion molecules, which are crucial for the survival and metastases of tumor cells. Elevated expression of CEACAM6 and SCGB3A2 was discovered in CSF-CTCs, which could serve as candidate biomarkers of LUAD-LM. We identified substantial heterogeneity in CSF-CTCs among LUAD-LM patients and within patient among individual cells. Cell-cycle gene expression profiles and the proportion of CTCs displaying mesenchymal and cancer stem cell properties also vary among patients. In addition, CSF-CTC transcriptome profiling identified one LM case as cancer of unknown primary site (CUP). Our results will shed light on the mechanism of LUAD-LM and provide a new direction of diagnostic test of LUAD-LM and CUP cases from CSF samples.

scholarly journals The single-cell transcriptional landscape of lung carcinoid tumors

2021 ◽  
Author(s):  
Philip Bischoff ◽  
Alexandra Trinks ◽  
Jennifer Wiederspahn ◽  
Benedikt Obermayer ◽  
Jan Patrick Pett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Carcinoid Tumor ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Carcinoid Tumors ◽  
Cellular Composition ◽  
Cell Gene Expression ◽  
Cell Gene ◽  
Lung Carcinoid

AbstractLung carcinoid tumors, also referred to as pulmonary neuroendocrine tumors or lung carcinoids, are rare neoplasms of the lung with a more favorable prognosis than other subtypes of lung cancer. Still, some patients suffer from relapsed disease and metastatic spread while no consensus treatment exists for metastasized carcinoids. Several recent single-cell studies have provided detailed insights into the cellular heterogeneity of more common lung cancers, such as adeno- and squamous cell carcinoma. However, the characteristics of lung carcinoids on the single-cell level are yet completely unknown.To study the cellular composition and single-cell gene expression profiles in lung carcinoids, we applied single-cell RNA sequencing to three lung carcinoid tumor samples and normal lung tissue. The single-cell transcriptomes of carcinoid tumor cells reflected intertumoral heterogeneity associated with clinicopathological features, such as tumor necrosis and proliferation index. The immune microenvironment was specifically enriched in noninflammatory monocyte-derived myeloid cells. Tumor-associated endothelial cells were characterized by distinct gene expression profiles. A spectrum of vascular smooth muscle cells and pericytes predominated the stromal microenvironment. We found a small proportion of myofibroblasts exhibiting features reminiscent of cancer-associated fibroblasts. Stromal and immune cells exhibited potential paracrine interactions which may shape the microenvironment via NOTCH, VEGF, TGFβ and JAK/STAT signaling. Moreover, single-cell gene signatures of pericytes and myofibroblasts demonstrated prognostic value in bulk gene expression data.Here, we provide first comprehensive insights into the cellular composition and single-cell gene expression profiles in lung carcinoids, demonstrating the non-inflammatory and vessel-rich nature of their tumor microenvironment, and outlining relevant intercellular interactions which could serve as future therapeutic targets.

scholarly journals Single-nucleus RNA-seq identifies Huntington disease astrocyte states

2019 ◽  
Author(s):  
Osama Al-Dalahmah ◽  
Alexander A Sosunov ◽  
A Shaik ◽  
Kenneth Ofori ◽  
Yang Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Huntington Disease ◽  
Expression Profiles ◽  
Lipid Synthesis ◽  
Gene Expression Profiles ◽  
Cag Repeats ◽  
Cell Gene Expression ◽  
Single Nucleus ◽  
Cell Gene

AbstractHuntington Disease (HD) is an inherited movement disorder caused by expanded CAG repeats in the Huntingtin gene. We have used single nucleus RNASeq (snRNASeq) to uncover cellular phenotypes that change in the disease, investigating single cell gene expression in cingulate cortex of patients with HD and comparing the gene expression to that of patients with no neurological disease. In this study, we focused on astrocytes, although we found significant gene expression differences in neurons, oligodendrocytes, and microglia as well. In particular, the gene expression profiles of astrocytes in HD showed multiple signatures, varying in phenotype from cells that had markedly upregulated metallothionein and heat shock genes, but had not completely lost the expression of genes associated with normal protoplasmic astrocytes, to astrocytes that had substantially upregulated GFAP and had lost expression of many normal protoplasmic astrocyte genes as well as metallothionein genes. When compared to astrocytes in control samples, astrocyte signatures in HD also showed downregulated expression of a number of genes, including several associated with protoplasmic astrocyte function and lipid synthesis. Thus, HD astrocytes appeared in variable transcriptional phenotypes, and could be divided into several different “states”, defined by patterns of gene expression. Ultimately, this study begins to fill the knowledge gap of single cell gene expression in HD and provide a more detailed understanding of the variation in changes in gene expression during astrocyte “reactions” to the disease.

scholarly journals Quantification of cell identity from single-cell gene expression profiles

2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Idan Efroni ◽  
Pui-Leng Ip ◽  
Tal Nawy ◽  
Alison Mello ◽  
Kenneth D Birnbaum
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Identity ◽  
Cell Gene Expression ◽  
Cell Gene

scholarly journals Single-Cell Gene Expression Profiles Define Self-Renewing, Pluripotent, and Lineage Primed States of Human Pluripotent Stem Cells

2014 ◽  
Vol 2 (6) ◽  
pp. 881-895 ◽  
Author(s):  
Shelley R. Hough ◽  
Matthew Thornton ◽  
Elizabeth Mason ◽  
Jessica C. Mar ◽  
Christine A. Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Human Pluripotent Stem Cells ◽  
Cell Gene Expression ◽  
Cell Gene

scholarly journals Diffusion pseudotime robustly reconstructs lineage branching

2016 ◽  
Author(s):  
Laleh Haghverdi ◽  
Maren Büttner ◽  
F. Alexander Wolf ◽  
Florian Buettner ◽  
Fabian J. Theis
Keyword(s):  
Gene Expression ◽  
Random Walks ◽  
Single Cell ◽  
Temporal Order ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Metastable States ◽  
Length Scales ◽  
Cell Gene Expression ◽  
Cell Gene

Single-cell gene expression profiles of differentiating cells encode their intrinsic latent temporal order. We describe an efficient way to robustly estimate this order according to a diffusion pseudotime, which measures transitions on all length scales between cells using diffusion-like random walks. This allows us to identify cells that undergo branching decisions or are in metastable states, and thereby genes differentially regulated at these states.

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