Genome Editing, single cell analysis and RNA profiling
Our work is mainly focused on the generation of gene Knockouts (KOs) and Knockins (KIs) in different cell types/cell lines. The KO and KI generation in human induced pluripotent stem cells (iPSCs) has become a main focus in the lab.
To generate KO and KI cells, we make use of the CRISPR/Cas system that has been evolved in bacteria to fight pathogenic bacteriophages. This system is based on a guide RNA (gRNA) and the Cas protein. While the gRNA guides the Cas protein to a specific gene locus, the Cas enzyme induces a DNA double-strand break. To induce the DNA strand break, the Cas enzyme additionally requires a short nucleotide sequence called protospacer adjacent motif (PAM) at the respective gene locus. The most frequently used Cas9 enzyme from Streptococcus pyogenes requires a NGG, while the Cas12a (Cpf1) enzyme from Prevotella and Francisella requires a TTTV. Erroneously repaired double-strand breaks by the host DNA damage repair machinery can cause the inactivation of the respective gene and potentially the death of the bacteriophage.
As it allows genome editing at almost every gene locus, the CRISPR/Cas system was adapted in recent years by the scientific field to generate KO and KI cells. The only requirement is a PAM present at the gene locus of interest. To generate a gene knockout, a gene-specific gRNA and the Cas protein need to be delivered into the cell. The error-prone repair machinery of the cell can subsequently cause mutations that can lead to the inactivation of the gene. In order to create a gene knock-in, a repair template (usually a single-stranded oligonucleotide) is delivered in addition to the gRNA and Cas protein that serves as a template for the repair machinery.
We have experience and expertise in using high efficiency CRISPR/Cas approaches to generate genetically modified cells and animals:
- gene knock-outs
- conditional knock-outs
- variant allele knock-ins
- transgene knock-ins
We provide advice and assistance to researchers on:
- the development of CRISPR/Cas9-based genome editing strategies
- the selection of suitable gRNAs
- the design of ssDNA repair templates
- the design and construction of DNA repair templates
- strategies for the genotyping and genetic characterization of genetically modified cells
- the establishment and maintenance of different cell lines
We are also constantly working to improve existing methods and to develop new approaches which will allow us to design more efficient and successful genome editing strategies, including CRISPR and TALEN.
For instance, we recently established a new selection method to isolate genetically modified cells which allows to select edited cells in few minutes.
Single cell sequencing and RNA profiling
Single cell sequencing allows the analysis of the sequence information from individual cells with optimized next-generation sequencing (NGS) technologies. Thus, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment.
This technology becomes particularly interesting when dealing with the effect of pollution on different cell types of the lung or the effect of UV light on different skin cell types.
For instance, single cell ATAC will be used in this context to:
- Discover cellular heterogeneity originating from epigenetic variability
- Better understand the gene regulatory networks that are upstream of gene expression
- Define cell types and states for lineage and developmental program tracing
- Accelerate biomarker discovery
The functional relevance of the expression/activation of identified target genes will be further investigated by using CRISPR knockout.
As part of the service, the Core Unit is also offering the Massive analysis of cDNA ends (MACE) which allows for accurate and reproducible transcriptome profiling without any PCR amplification bias.
Improving Genome Engineering tools
Development of in vitro human lung models from cell lines and primary cells
The project is related to the research field 1 of the IUF in cooperation with the research groups of PD Dr. Klaus Unfried und Dr. Roel Schins. The project aims to develop in vitro human lung models from cell lines and primary cells that are suitable to resemble the human lung physiology and morphology. Therefore, human lung-derived cells (bronchial, small airway and alveolar cells) are cultured at the air-liquid interphase to induce cell differentiation and 3D cell expansion into a pseudostratified epithelium cell layer. Co-cultures of different cell types (epithelial cells, endothelial cells, macrophages) could be used to resemble the complex cellular interplay between different cell types in that tissue. The air-liquid interphase culture technique mimics the in vivo situation where lung epithelial and alveolar cells are exposed to the airway at the apical side but are supplied with nutrients from the basolateral side. These lung models will be used to test the potential harmful effects of airborne particles and nanoparticles. For this purpose, a nanoparticle generator in combination with an automated exposure station from the German company Vitrocell® is available. The particles are generated by spark ablation with a small aerodynamic diameter and a high efficiency and guided into the exposure station. The exposure station relies on the continuous flow technique to guarantee a highly standardized and reproducible experimental setup for particle exposure to cells cultured at the air-liquid interphase.
An active collaboration with the Institute for Energy and Environmental Technology (IUTA) in Duisburg aims to characterize the generated particles from the particle generator for size distribution and particle properties.
Bioengineering the microanatomy of human skin
The main aim of this project is to establish a new laboratory for the development of complex long term cultivable human three-dimensional skin models. The project will start off by expanding the range of skin models already implemented in house (from Boukamp´s group) in order to use them for a variety of applications including complementation with immune cells.
This novel immunocompetent 3D human skin model, once fully optimised, could then be used as a platform in order to investigate how environmental factors (ultraviolet radiation, air pollution and chemicals in general) affect the physiology and pathophysiology of the skin, supporting in this way other research groups at IUF.