Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells

Alda-Catalinas, Celia; Eckersley-Maslin, Mélanie; Reik, Wolf

doi:10.1016/j.xpro.2021.100426

Cited by 3 publications

(1 citation statement)

References 7 publications

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“…3' gene expression libraries (v3) were prepared following manufacturer's instructions (10X Genomics, PN-1000075). gRNA amplicon libraries were generated from amplified cDNA following the protocol described in (Alda-Catalinas et al 2021). Gene expression and gRNA libraries were QCed by tapestation (Agilent) and quantified by Qubit (Thermo Fisher, Q32851) and KAPA qPCR (Kapa Biosystems, KK4824).…”

Section: Pooled Crispri Screenmentioning

confidence: 99%

Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

Alda-Catalinas

Ibarra-Soria

Flouri

et al. 2023

Preprint

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Drug targets with human genetic evidence are expected to increase clinical success by at least two-fold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of early drug discovery. A key issue is that, currently, the vast majority of complex disease associations cannot be cleanly mapped to a gene. Immune disease-associated variants are enriched within regulatory elements, such as distal enhancers, found in T cell-specific open chromatin regions. To identify the genes and thus the molecular programs modulated by these regulatory elements, we developed a CRISPRi-based single-cell functional screening approach in primary human CD4+ T cells. Our pipeline enables the interrogation of transcriptomic changes induced by the perturbation of regulatory elements at scale. We first optimised a highly efficient CRISPRi protocol in primary human CD4+ T cells via CROPseq vectors. Subsequently, we performed a proof-of-concept screen targeting 45 non-coding regulatory elements and 35 transcription start sites and profiled approximately 250,000 CD4+ T cell single-cell transcriptomes. We developed a bespoke analytical pipeline for element-to-gene (E2G) mapping and demonstrate that our method can identify both previously annotated and novel E2G links. Lastly, we integrated genetic association data for immune-related traits and demonstrate how our platform can aid in the identification of effector genes for GWAS loci.

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Section: Pooled Crispri Screenmentioning

confidence: 99%

Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

Alda-Catalinas

Ibarra-Soria

Flouri

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

Alda-Catalinas,

Ibarra-Soria,

Flouri

et al. 2024

Genome Biol

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Background Drug targets with genetic evidence are expected to increase clinical success by at least twofold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of drug discovery. A key issue is that the vast majority of complex disease associations cannot be cleanly mapped to a gene. Immune disease-associated variants are enriched within regulatory elements found in T-cell-specific open chromatin regions. Results To identify genes and molecular programs modulated by these regulatory elements, we develop a CRISPRi-based single-cell functional screening approach in primary human T cells. Our pipeline enables the interrogation of transcriptomic changes induced by the perturbation of regulatory elements at scale. We first optimize an efficient CRISPRi protocol in primary CD4+ T cells via CROPseq vectors. Subsequently, we perform a screen targeting 45 non-coding regulatory elements and 35 transcription start sites and profile approximately 250,000 T -cell single-cell transcriptomes. We develop a bespoke analytical pipeline for element-to-gene (E2G) mapping and demonstrate that our method can identify both previously annotated and novel E2G links. Lastly, we integrate genetic association data for immune-related traits and demonstrate how our platform can aid in the identification of effector genes for GWAS loci. Conclusions We describe “primary T cell crisprQTL” — a scalable, single-cell functional genomics approach for mapping regulatory elements to genes in primary human T cells. We show how this framework can facilitate the interrogation of immune disease GWAS hits and propose that the combination of experimental and QTL-based techniques is likely to address the variant-to-function problem.

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Single-cell Technology in Stem Cell Research

Golchin,

Shams,

Moradi

et al. 2025

CSCR

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Single-cell technology (SCT), which enables the examination of the fundamental units comprising biological organs, tissues, and cells, has emerged as a powerful tool, particularly in the field of biology, with a profound impact on stem cell research. This innovative technology opens new pathways for acquiring cell-specific data and gaining insights into the molecular pathways governing organ function and biology. SCT is not only frequently used to explore rare and diverse cell types, including stem cells, but it also unveils the intricacies of cellular diversity and dynamics. This perspective, crucial for advancing stem cell research, facilitates non-invasive analyses of molecular dynamics and cellular functions over time. Despite numerous investigations into potential stem cell therapies for genetic disorders, degenerative conditions, and severe injuries, the number of approved stem cell-based treatments remains limited. This limitation is attributed to the various heterogeneities present among stem cell sources, hindering their widespread clinical utilization. Furthermore, stem cell research is intimately connected with cutting-edge technologies, such as microfluidic organoids, CRISPR technology, and cell/tissue engineering. Each strategy developed to overcome the constraints of stem cell research has the potential to significantly impact advanced stem cell therapies. Drawing from the advantages and progress achieved through SCT-based approaches, this study aims to provide an overview of the advancements and concepts associated with the utilization of SCT in stem cell research and its related fields.

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Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells

Cited by 3 publications

References 7 publications

Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens

Single-cell Technology in Stem Cell Research

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