Understanding how genetic variation and mutations lead to the onset of human diseases is an important question in biomedical research and the basis for developing therapeutic interventions.
We are using the CRISPR/Cas nuclease technology in human induced pluripotent stem cells (hiPSCs) as central technology to investigate human genetic disease mechanisms. hiPSCs are cell lines can be indefinitely propagated but also differentiated in vitro into many cell types of the human body.
The research group on hiPSC -based disease modeling is setting up a technology platform enabling the engineering, differentiation and phenotyping of hiPSCs. The lab is presently focusing on the optimization of CRISPR/Cas induced mutagenesis in hiPSCs and their directed differentiation into dopaminergic neurons. The first topic requires the efficient delivery and action of Cas9 nuclease, gene specific sgRNAs and customized DNA repair templates molecules in hiPSCs. In the second working area we are defining the optimal combination of external and internal cues specifying the differentiation of hiPSCs into dopaminergic neurons to study genetic lesions leading to the loss of these cells in Parkinson`s disease.
Gene editing using the CRISPR/Cas nuclease system
Programmable nucleases are used for editing the sequence of target genes by breakup of the DNA at preselected positions. The latest generation of nucleases is provided by the CRISPR/Cas9 bacterial defense system that uses short, single guide (sg) RNAs for DNA sequence recognition and can be programmed towards new targets by adaption of the sgRNA first 20 nucleotides. sgRNAs are bound by the generic nuclease Cas9 and guide the complex to the complementary DNA sequence, followed by the induction of a double-strand break (DSB). DSBs are fixed in mammalian cells mainly by the NHEJ repair pathway which reconnects open DNA ends imprecisely and frequently leads to the loss of multiple base pairs at the target site.
Such small, randomly sized deletions are often used for the creation of frame shift mutations within coding regions leading to gene inactivation (knockout). The generation of precise modifications such as nucleotide replacements by gene targeting requires the alternative pathway of homology directed repair (HDR), able to read new sequences from externally delivered, DNA templates into the DSB site (knock-in). Only a small fraction of DSBs in mammalian cells is repaired by HDR, making precise gene editing inefficient. Using reporter constructs indicating the ratio of NHEJ or HDR for the repair of DSBs we found that the suppression of the NHEJ enzyme DNA ligase IV by a small molecule inhibitor or by adenoviral proteins is sufficient for stimulating DSB repair by HDR. This intervention facilitates the introduction of knock-in sequence modifications in cell lines and mice, as required for the precise modelling of patient derived mutations. Furthermore, this and ongoing work on the active promotion of HDR provides a basis for the future application of the CRISPR/Cas system in somatic gene therapy.
Studying disease pathways by gene editing in human IPS cells
Human induced pluripotent stem cells (hiPSCs) are early embryonic cell lines which can be established by the differentation of skin fibroblasts through the expression of reprogramming factors. hiPSCs can be indefinitely propagated but also differentiated in vitro into major cell types such as neurons or cardiomyocytes.
By combining he hiPS cell and CRISPR/Cas nuclease technologies biomedical research can investigate human genotype/ phenotype relations. The nuclease-assisted assembly of genotypes enables for example to introduce Parkinson`s isease risk alleles into `healthy´ genetic backgrounds or the correction of such alleles in patient-derived hiPS cells. For phenotypic analysis mutant and control cells are converted using lineagespecific transcription factors into differentiated cells, such as dopaminergic neurons. The differentiated cultures can be studied by live cell imaging for disease associated phenotypes, ultimately building a relational map of genotypes, envirotypes and phenotypes.
Since fall 2014 we focus on the set up of this hiPS cell technology platform for the simultaneous modification of up to three genes (multiplex engineering) by optimizing the delivery of Cas9, sgRNAs and HDR templates into hiPSCs and for the differentiation of hiPSCs into dopaminergic neurons. Presently we achieve in up to 30% of transfected hiPSCs the mutagenesis of two genes and can differentiate 1/3 of the cells into dopaminergic neurons. To support the analysis of cultures by live cell imaging we use fluorescent reporters by knockin into marker genes. In addition, we aim for extending the utility of the hIPSC technology platform by establishing CRISPR/Cas based conditional, i.e. inducible and cell type-specific gene knockout in differentiated cells and the three dimensional differentiation of hiPSCs into organoids which recapitulate aspects of fetal brain development.