Abstract: SA-OR029
In Vivo Pooled CRISPR/Cas9 Screening with Single Cell Transcriptome Readout to Analyze Genetic Modifications in Primary Murine T Cells in Crescentic Glomerulonephritis
Session Information
- ANCA It Is
November 09, 2019 | Location: 207, Walter E. Washington Convention Center
Abstract Time: 06:06 PM - 06:18 PM
Category: Glomerular Diseases
- 1202 Glomerular Diseases: Immunology and Inflammation
Authors
- Enk, Leon U. B., UMC Hamburg-Eppendorf, Hamburg, Germany
- Riecken, Kristoffer, UMC Hamburg-Eppendorf, Hamburg, Germany
- Kilian, Christoph, UMC Hamburg-Eppendorf, Hamburg, Germany
- Paust, Hans-Joachim, UMC Hamburg-Eppendorf, Hamburg, Germany
- Kapffer, Sonja, UMC Hamburg-Eppendorf, Hamburg, Germany
- Borchers, Alina, UMC Hamburg-Eppendorf, Hamburg, Germany
- Hellmig, Malte, UMC Hamburg-Eppendorf, Hamburg, Germany
- Bartsch, Patricia, UMC Hamburg-Eppendorf, Hamburg, Germany
- Zinke, Michael, UMC Hamburg-Eppendorf, Hamburg, Germany
- Panzer, Ulf, UMC Hamburg-Eppendorf, Hamburg, Germany
- Fehse, Boris, UMC Hamburg-Eppendorf, Hamburg, Germany
- Krebs, Christian F., UMC Hamburg-Eppendorf, Hamburg, Germany
Group or Team Name
- AG Krebs
Background
CRISPR/Cas9-based screening approaches usually lack the analysis of transcriptional perturbations caused by genetic modifications. In contrast, investigation of individual genetic modifications is time consuming and expensive. Therefore, CRISPR droplet sequencing (CROP-seq) has been developed to perform in vitro pooled CRISPR screening with single-cell transcriptome readout. However, examining changes in the cellular phenotype due to genetic modifications is strongly dependent on the local micro-environment. To address this, we have established an approach to target renal T cells in a mouse model of crescentic glomerulonephritis (cGN) to investigate transcriptional perturbations at the single cell level under the influence of a genetic modification in the context of renal autoimmunity.
Methods
CD4+ T cells from Il17aCre x Cas9Gfp mice were polarized towards a Th17 phenotype and transduced with lentiviral vectors encoding BFP and individual guide-RNAs (gRNAs) targeting IL17A, CD2 or scrambled-control. In another set of experiments, cells were transduced with a pool of gRNAs targeting 28 genes. Subsequently, cells were transferred into Rag1-deficient mice and experimental cGN (nephrotoxic nephritis) was induced. At day 10 renal T cells were analyzed by flow cytometry for protein expression in case of individual gRNAs or FACS-sorted and subjected to Single Cell RNA-seq in the pooled approach.
Results
Using individual gRNAs, we identified a highly significant reduction of IL17A producing cells after treatment with anti-Il17a gRNA and of cells' surface expression of CD2 after treatment with anti-CD2 gRNA, while scrambled-gRNA had no effect on IL-17A and CD2. Furthermore, we show that a pool of gRNAs targeting 28 genes can be used to identify individual transcriptional profiles of cells with defined gene knockdowns.
Conclusion
In vivo CROP-seq is a new technique that enables CRISPR-screens with single cell transcriptome read-out in experimental mouse models for the first time. This approach might be of major interest for the investigation of genetic modifications not only in the context of T cell-driven autoimmunity but also in a broader range of applications.
Funding
- Government Support - Non-U.S.