CRISPR/Cas based tissue engineering

Application of CRISPR/Cas based technologies are revolutionizing biology and are laying ground for various new approaches of synthetic biology. In its simplest form the Cas9 nuclease is guided by a single guide RNA (sgRNA) to a specific site in the genome, where it introduces a double strand break (DSBs). Thus, the technology is used to change eukaryotic genomes in a programmed way. Here, we want to establish a completely new application of the system: CRISPR/Cas based tissue engineering. The Puchta group at the KIT was able to show that the Cas9 nuclease from Staphylococcus aureus (SaCas9) is a much more efficient tool for DSB induction in plants than the widely used enzyme from Streptococcus pyogenes (SpCas9). A surprising discovery was that if a single sgRNA is used to target SaCas9 at highly repetitive sequences in the genome, like the rDNA genes or centromeric repeats, the repair machinery is overwhelmed and cell death can be induced. Thus, SaCas9 should be useable as a novel tool for killing defined classes of cells (“CRISPR-Kill”). The aim of the suggested project is to test if CRISPR-Kill is able to eliminate specific stem cell types out of more complex organisms or organs by modulating its expression. Thus, artificial developmental switches or new combinations of cell types for biochemical synthesis should come into reach. The potential of the technology to reprogram root and shoot development in plants should be elucidated based on the unique expertise of the Wolf group at the UHD. The group has setup sophisticated tools for microscopically monitoring microscopically root and shoot development in combination with cell type specific expression systems. Depending on the promoters used for SaCas9 expression, it should be possible to eliminate different stem cell types at will during growth and study developmental and biochemical consequences. The general potential of the technology reaches far beyond plants and might be useful for the production of synthetic organs.