An inducible CRISPR/dCAS9 strategy for directed differentiation of pluripotent stem cells

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Cell therapy such as haematopoietic stem cell transplantation and red blood cell or platelet transfusion are used to treat many diseases of the blood and immune systems but these treatments are highly dependent on a limited supply of healthy donors. Also, despite extensive screening of donated blood, there is always a chance that the patient could get an infection from the donated cells. To solve these problems there has been a lot of effort to generate blood cells in the laboratory from a limitless and infection-free source. One such source is human pluripotent stem cells (hPSCs) that can be grown indefinitely in the lab as stem cells and then, under defined conditions, can be differentiated into any cells type including blood cells. However, the efficiency of this process is very low and it has not been possible to generate fully functional blood cells. Several studies have shown that the production and function of specific cell types can be improved by "directing" the cells into specific cell types by activating the expression of factors that act as molecular switches to turn on the expression of genes that are required for a particular cell function. These studies have depended on introducing transgenes into the cells using plasmid or lentiviral vectors but they result in abnormally high levels of gene expression. We propose to use a novel strategy that results in the activation of the cells' own genetic networks and results in more physiological levels of gene expression.We will first compare the gene profile of hPSC- with adult-derived blood cells and this will identify the genetic switches required for the production of fully functional adult-like blood cells as well as novel markers that can be used to track the production process. We will then test whether activation of these genetic switches in differentiating hPSCs will result in the improved production of blood cells from hPSCs. To do this we will use a novel synthetic biology strategy whereby our chosen factors can be turned on using small molecular tools known as guide RNAs (gRNAs). gRNAs are designed to bind to the region of the genome that regulates the expression of a particular gene(s) and, together with a protein complex, known as CAS9-SAM, the expression of the gene can be activated. One of the unique aspects of our proposal is that we have designed and tested an hPSC line in which activation of the protein complex can be induced upon addition of a drug. This novel iSAM strategy represents a significant advance in the field as it makes the approach amenable to the activation of multiple factors at once which is likely to be required for the generation of fully functional blood cells. We will first use our iSAM strategy to activate single and combinations of genetic factors and monitor their effects on the production of blood cells. This will generate an experimental pipeline that will then allow us to screen all the genes in the genome in one single experiment. Cells that we produce by programming with genetic factors will be tested using methods such as flow cytometry to assess the presence of markers on the cell surface, colony-forming assays to monitor blood cell progenitors and single cell RNA sequencing to analyse the transcriptional consequences of the programming process. This project will provide a better understanding of genetic factor programming and could provide a route to producing cells for the treatment of patients with blood cell disorders. Our unique iPSC line carrying the iSAM complex will be shared with researchers studying the programming and production of other therapeutic cell types such as dopaminergic neurons to treat Parkinsons disease or pancreatic beta cells for Diabetes. Our strategy offers significant advantages over the classical transgenic technologies that are fraught with technical difficulties such as gene silencing and insertional mutagenesis that would raise significant safety concerns in the clinic.