Genome Engineering and Model Development lab (GEMD)
Research profile
The main task of the core unit is to further develop existing and to establish new organotypic human cultures, to use cutting-edge techniques to manipulate the genome of these models as well as the generation of genetically modified animal models (e.g. C. elegans). In the medium term, the development of the following models is planned for research field 1: lung models based on primary cells which allow the exposure with airborne particles and nanoparticles in the air flow; for research field 3: improvement of existing neurosphere models and development of brain organoids; for research field 2 and 4: long-term cultivable (up to 6 months), human, three dimensional skin models from fibroblasts and keratinocytes.
Projects
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.
Selected publications
El-Brolosy
M, Rossi A, Kontarakis Z, Kuenne C, Günther S, Fukuda N, Khrievono K,
Boezio G, Takacs C, Lai SL, Fukuda R, Gerri C, Giraldez J, Didier YR,
Stainier DYR: Genetic compensation triggered by mutant mRNA degradation. Nature 2019. (Highlighted
by Jan Philip Junker Detouring the roadblocks in gene expression) [pubmed]
Rossi
A, Gauvrit S, Stainier DY: Regulation of Vegf signaling by natural and
synthetic ligands. Blood 128(19): 2359-2366,
2016. [pubmed] (Highlighted by Tatiana Byzova “Fishing” out the real VEGFs)
Rossi
A, Kontarakis Z, Gerri G, Nolte H, Hölper S, Krüger M, Stainier DY: Genetic
compensation induced by deleterious mutations but not gene knockdowns. Nature 524(7564):
230-233, 2015. [pubmed]
Stainier
DY, Kontarakis Z, Rossi A: Making sense of anti-sense data. Dev
Cell 32(1): 7-8, 2015. [pubmed]
Rossi
A, Moritz T, Ratelade J, Verkman AS: Super-resolution imaging of aquaporin-4
orthogonal array of particles in cell membranes. J Cell Sci 125(18): 4405-4412, 2012. [pubmed]
Further publications of Dr. Rossi on google scholar.