Hornung Lab - Research

Genome engineering in immune cells

A series of game-changing developments have revolutionized the field of genome engineering within the past decade. With the discovery and of the CRISPR/Cas system, it has now become possible to alter the genomic architecture of a wide range of different cell types in a user-defined fashion. Within the laboratory, we have been implementing CRISPR/Cas-based genome engineering technologies since their initial description to study innate immune sensing and signaling cascades. To this end, we have set up various CRISPR/Cas-based workflows in the lab:

Generating knock-out cells

For many projects, we are primarily interested in the generation of knockout cell lines. To this end, we target a critical exon of the gene of interest and aim at the disruption of the reading frame by introducing indel mutations. This can lead to the introduction of premature stop codons and as such degradation of the cognate mRNA due to nonsense mediated mRNA decay or alternatively to truncated proteins.

For various cell lines, we have set up workflows that allow us to obtain single-cell clones following the gene-targeting procedure. This is achieved by limiting dilution cloning, which we have automated to a large degree on our liquid handling workstations. Following automated clone analysis, single-cell clones are picked and duplicated. One duplicate is lysed to perform a locus-specific PCR and a subsequent second PCR that attaches barcodes and sequencing adapters. Finally, all PCR products are pooled and subjected to deep sequencing. Sequencing reads are then analyzed to identify cell clones in which all alleles are disrupted by a frame-shifting indel mutation.

For knock-out approaches, we have generated a plasmid-based genome-wide gRNA library that currently covers 85,45 % of the human protein-coding genome. This library is available as 17155 individually arrayed constructs that can be transduced in a lentiviral format. This resource allows us to conduct mid-scale, reverse-genetic screening approaches in various cell types with little preparatory time.

Apart from plasmid- or lentivirus-based systems, we have also adopted ribonucleoprotein (RNP) based systems, in which a recombinant Cas9 protein is delivered into cells in complex with a synthetic guide RNA. Apart from increasing targeting efficiency, this approach allows us to target cells that are usually not amenable to plasmid or lentivirus delivery (e.g. primary human T cells or hematopoietic stem cells).

Forward genetic screens

Next to hypothesis-driven knock-out projects, we also conduct forward genetic screens, in which we select cells for their phenotype of interest. For this, we have established the use of genome-wide libraries targeting the murine and the human genome. Screens selecting for cell survival, but also for complex phenotypes (e.g. certain cell death phenotypes or surface markers) are being conducted.

 

Recent publications:

CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.
Schmid-Burgk JL, Höning K, Ebert TS, Hornung V.
Nat Commun. 2016 Jul 28;7:12338. doi: 10.1038/ncomms12338. PubMed

Designer Nuclease-Mediated Generation of Knockout THP1 Cells.
Schmidt T, Schmid-Burgk JL, Ebert TS, Gaidt MM, Hornung V.
Methods Mol Biol. 2016;1338:261-72. doi: 10.1007/978-1-4939-2932-0_19. PubMed

Synthesis of an arrayed sgRNA library targeting the human genome.
Schmidt T, Schmid-Burgk JL, Hornung V.
Sci Rep. 2015 Oct 8;5:14987. doi: 10.1038/srep14987. PubMed

Ligation-independent cloning (LIC) assembly of TALEN genes.
Schmid-Burgk JL, Schmidt T, Hornung V.
Methods Mol Biol. 2015;1239:161-9. doi: 10.1007/978-1-4939-1862-1_8. PubMed

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines.
Schmid-Burgk JL, Schmidt T, Gaidt MM, Pelka K, Latz E, Ebert TS, Hornung V.
Genome Res. 2014 Oct;24(10):1719-23. doi: 10.1101/gr.176701.114. Epub 2014 Sep 3. PubMed

A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes.
Schmid-Burgk JL, Schmidt T, Kaiser V, Höning K, Hornung V.
Nat Biotechnol. 2013 Jan;31(1):76-81. PubMed