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Extensive mapping of an innate immune network with CRISPR.

Aregger M, Hart T, Moffat J - Mol. Syst. Biol. (2015)

Bottom Line: The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking.Parnas et al (2015) apply this screening technology to mouse bone marrow‐derived dendritic cells in order to study the regulation of the immune response triggered by PAMPs.Through integrated analysis of gene knockouts in conjunction with changes in protein and mRNA expression, CRISPR screens are facilitating dissection of immune regulatory networks at unprecedented resolution.

View Article: PubMed Central - PubMed

Affiliation: Donnelly Centre, University of Toronto, Toronto, ON, Canada.

ABSTRACT
The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking. Parnas et al (2015) apply this screening technology to mouse bone marrow‐derived dendritic cells in order to study the regulation of the immune response triggered by PAMPs. Through integrated analysis of gene knockouts in conjunction with changes in protein and mRNA expression, CRISPR screens are facilitating dissection of immune regulatory networks at unprecedented resolution.

No MeSH data available.


A genome-wide pooled CRISPR screen in mouse primary immune cells to dissect regulatory networksDesign of the genome-wide primary and secondary screens by FACS sorting, deep sequencing and subsequent assignment of hits to functional modules based on their effect on RNA and protein expression to map immune regulatory networks [heatmap adapted from Parnas et al (2015), ©CellPress].
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fig01: A genome-wide pooled CRISPR screen in mouse primary immune cells to dissect regulatory networksDesign of the genome-wide primary and secondary screens by FACS sorting, deep sequencing and subsequent assignment of hits to functional modules based on their effect on RNA and protein expression to map immune regulatory networks [heatmap adapted from Parnas et al (2015), ©CellPress].

Mentions: Now this technology has been used to deconstruct a complex biological process, the first application of CRISPR-Cas9 to a systems-level biological question in mammalian cells. Parnas and colleagues report screening primary mouse bone marrow-derived dendritic cells (DCs), where they dissect the regulatory network associated with induction of tumour necrosis factor or TNF (Parnas et al, 2015). The authors use bone marrow-derived DCs isolated from Cas9-expressing transgenic mice to study the innate immune response to lipopolysaccharide (LPS) through Toll-like receptors (TLR) (Fig1). Parnas et al (2015) performed a genome-wide pooled CRISPR screen on these ex vivo cells and, after activation with LPS, sorted the cells based on high or low protein expression of the inflammatory cytokine TNF. The primary screen identified most known regulators of TNF expression and TLR4 signalling, as well as novel hits that were validated with individual single-guide RNAs (sgRNAs). This analysis was followed by a deeper secondary screen comprising the top ranked 2,569 genes and revealed additional regulators of TNF expression with greater sensitivity.


Extensive mapping of an innate immune network with CRISPR.

Aregger M, Hart T, Moffat J - Mol. Syst. Biol. (2015)

A genome-wide pooled CRISPR screen in mouse primary immune cells to dissect regulatory networksDesign of the genome-wide primary and secondary screens by FACS sorting, deep sequencing and subsequent assignment of hits to functional modules based on their effect on RNA and protein expression to map immune regulatory networks [heatmap adapted from Parnas et al (2015), ©CellPress].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4547849&req=5

fig01: A genome-wide pooled CRISPR screen in mouse primary immune cells to dissect regulatory networksDesign of the genome-wide primary and secondary screens by FACS sorting, deep sequencing and subsequent assignment of hits to functional modules based on their effect on RNA and protein expression to map immune regulatory networks [heatmap adapted from Parnas et al (2015), ©CellPress].
Mentions: Now this technology has been used to deconstruct a complex biological process, the first application of CRISPR-Cas9 to a systems-level biological question in mammalian cells. Parnas and colleagues report screening primary mouse bone marrow-derived dendritic cells (DCs), where they dissect the regulatory network associated with induction of tumour necrosis factor or TNF (Parnas et al, 2015). The authors use bone marrow-derived DCs isolated from Cas9-expressing transgenic mice to study the innate immune response to lipopolysaccharide (LPS) through Toll-like receptors (TLR) (Fig1). Parnas et al (2015) performed a genome-wide pooled CRISPR screen on these ex vivo cells and, after activation with LPS, sorted the cells based on high or low protein expression of the inflammatory cytokine TNF. The primary screen identified most known regulators of TNF expression and TLR4 signalling, as well as novel hits that were validated with individual single-guide RNAs (sgRNAs). This analysis was followed by a deeper secondary screen comprising the top ranked 2,569 genes and revealed additional regulators of TNF expression with greater sensitivity.

Bottom Line: The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking.Parnas et al (2015) apply this screening technology to mouse bone marrow‐derived dendritic cells in order to study the regulation of the immune response triggered by PAMPs.Through integrated analysis of gene knockouts in conjunction with changes in protein and mRNA expression, CRISPR screens are facilitating dissection of immune regulatory networks at unprecedented resolution.

View Article: PubMed Central - PubMed

Affiliation: Donnelly Centre, University of Toronto, Toronto, ON, Canada.

ABSTRACT
The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking. Parnas et al (2015) apply this screening technology to mouse bone marrow‐derived dendritic cells in order to study the regulation of the immune response triggered by PAMPs. Through integrated analysis of gene knockouts in conjunction with changes in protein and mRNA expression, CRISPR screens are facilitating dissection of immune regulatory networks at unprecedented resolution.

No MeSH data available.