Team: Aleksandar Rakovic, PhD (group leader); Victor Krajka (PhD student); Britta Meier (research technologist)
The emerging CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) technology has been used to rapidly, easily and efficiently modify endogenous genes and genomic regions in a wide variety of different cell types and in organisms that have traditionally been challenging to manipulate genetically.
The system comprises of i) a short synthetic RNA (gRNA) composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified and ii) the endonuclease Cas9. Upon recognition of the genomic target sequence by the gRNA, Cas9 cleaves each of the targeted DNA strands.
The resulting DNA breakages will be repaired by the error-prone non-homologous end joining (NHEJ) pathway resulting in random insertions or deletions (InDels) inducing the frame shift and finally knocking out the targeted gene. Alternatively, Cas9-mediated DNA cleavages can be repaired by the homologous recombination (HR). By providing a donor DNAs one can introduce desired mutations or correct the existing ones genome wide.
The CRISPR/Cas9 genome editing platform is fully established within the Dr. Rakovic’s research group “Molecular mechanisms of Parkinson’s disease” at the Institute of Neurogenetics. His group has already successfully established ~20 CRISPR/Cas9-derived models of Parkinson’s disease and Dystonia using induced pluripotent stem cells (iPSC) and commonly used cell lines.
The CRISPR-Cas9 system is a game changing technology for precise and efficient alterations in genome sequence and gene expression. The accessibility of this novel genome editing technology will fundamentally improve and influence biological/medical research in the elucidation of molecular mechanisms as well as the development novel molecular therapeutics for human disease.
Figure 1. (A) Generation of Parkin, PINK1, and DJ1 knockout isogenic neuroblastoma lines using CRISPR/Cas9. Western blot analysis using antibodies against Parkin, PINK1, and DJ1 shows complete knockout of targeted genes. (B) CRISPR/Cas9-mediated removal of a large intronic insertion (SVA) in intron 32 of the Taf1 gene in iPSC derived from the XDP patient. PCR using “outer” primers seqF and seqR shows correction in two (clones 2 and 3) out of five analyzed clones.
Team: Philip Seibler, PhD (group leader); Philipp Capetian, MD (group leader); Karen Grütz (PhD student); Franca Vulinovic (PhD student); Franziska Rudolph (research technologist); Karen Rieck (research technologist); Eva-Maria Bernhardi (student)
Induced pluripotent stem cells (iPSCs) have gained increasing importance for stem cell-based disease modeling whereby tissue from a patient is reprogrammed to pluripotency (iPSC) and then differentiated into a cell type closely resembling the tissue bearing the brunt of disease. We use iPSC technology to functionally examine disease pathology in patient-derived, biologically relevant cells. The tasks of our “iPSC platform” are to undertake the reprogramming, biobanking, and differentiation required in the context of various projects. To reprogram patient skin fibroblasts into iPSCs, we employ Sendai virus transduction to deliver the four reprogramming factors OCT4, SOX2, KLF4, and cMYC by using a commercially available kit (CytoTune-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher Scientific Inc). Sendai virus is a non-integrating RNA virus that produces iPSC clones with high efficiency and of best quality, as has been shown by us and others. Stringent quality control steps established in our laboratory are applied to ensure that the iPSC lines generated are free of karyotypic abnormalities and demonstrate robust differentiation potential. All iPSC lines are cultured under feeder-free conditions and a basic stock of cryovials of each iPSC line is generated for dissemination. For the neurological movement disorders Parkinson disease and dystonia, we focus on dopaminergic neurons. Based on our previous work1-5, we differentiate iPSCs into midbrain dopaminergic neurons. The underlying protocol6 ventralizes the iPSCs into a midbrain floor plate phenotype first (the floor plate being a key organizer of the central nervous system, where the midbrain dopaminergic neurons originate). This is accomplished by using strong bone morphogenic protein inhibition coupled with Sonic Hedgehog and Wnt pathway activation to induce the floor plate-like phenotype. Neural induction is performed by adding numerous recombinant proteins and small molecules at various time points. After 75 days of differentiation, there is a cell culture consisting of more than 90% neurons, of which approximately 40% represent electrophysiologically active dopaminergic neurons based on whole-cell recordings and the expression of key markers tyrosine hydroxylase (marker for dopaminergic cells), TUJ1 (neuronal marker), and FOXA2 (midbrain marker).
Seibler P, Graziotto J, Jeong H, et al. Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells. J. Neurosci. 2011;31(16):5970–5976.
Rakovic A, Shurkewitsch K, Seibler P, et al. Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)-dependent ubiquitination of endogenous Parkin attenuates mitophagy: study in human primary fibroblasts and induced pluripotent stem cell-derived neurons. J. Biol. Chem. 2013;288(4):2223–2237.
Morais VA, Haddad D, Craessaerts K, et al. PINK1 loss-of-function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science 2014;344(6180):203–207.
Erogullari A, Hollstein R, Seibler P, et al. THAP1, the gene mutated in DYT6 dystonia, autoregulates its own expression. Biochim Biophys Acta. 2014;1839(11):1196-204.
Munsie LN, Milnerwood AJ, Seibler P, et al. Retromer-dependent neurotransmitter receptor trafficking to synapses is altered by the Parkinson’s disease VPS35 mutation p.D620N. Hum. Mol. Genet. 2015;24(6):1691–1703.
Kriks S, Shim J-W, Piao J, et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 2011;480(7378):547–551.