Exploring Early Human Development and Regenerative Medicine Utilizing the CRISPR/Cas Gene Editing System
The development of the CRISPR/Cas technology has paved the way for an accurate and readily available genome engineering tool. Guided by a small RNA molecule, Cas9 is localized to the genomic region of interest where it can perform a double-strand break. There are two main repair pathways activated upon DNA damage: Homology-Directed Repair (HDR) and Non-Homologous End-Joining (NHEJ). The former pathway relies on homology, either endogenous or exogenous, to repair the damage in a correcting manner whereas the latter, NHEJ, generates insertions or deletions (indels) at the break site. Indels generating frameshift mutations prevent normal protein translation usually by creating premature stop codon, making it possible to study cellular mechanisms and functions in the absence of a protein.
I am currently utilizing the CRISPR/Cas system to study loss-of-function in different contexts of development and regenerative medicine. These projects focus on, I) human X chromosome inactivation and II) reducing immunogenicity of hESC-derived cell types.
I. Human X chromosome inactivation
Different species have developed various forms of X chromosome dosage compensation, e.g. female preimplantation mouse embryos inactivate the paternal X chromosome at the four cell-stage, which is later re-activated in the inner cell mass of the blastocyst at which time random X chromosome inactivation follows. Human preimplantation embryos do not exhibit the same pattern of dosage compensation and although some genes have been previously identified as necessary for X chromosome inactivation (XCI) in mouse, their function in human dosage compensation is not as well-established. We will utilize the CRISPR/Cas system to study loss-of-function of selected genes to learn more about early human development.
II. Reducing immunogenicity of hESC-derived cell types
Immune-mediated rejection of allogenic transplants is an ongoing challenge with few long-term sustainable solutions. These transplantations rely on donor-host HLA matching to limit rejection of the transplanted tissue. Our approach relies on circumventing this response by modifying hESCs using the CRISPR/Cas system to reduce their immunological properties.