METHOD TO BIOENGINEER DESIGNER RED BLOOD CELLS USING GENE EDITING AND STEM CELL METHODOLOGIES

    公开(公告)号:US20200332259A1

    公开(公告)日:2020-10-22

    申请号:US16913741

    申请日:2020-06-26

    Abstract: A method of creating cells expressing specific red blood cell antigens is disclosed. In one embodiment, the method comprises the steps of (a) combining one or more guide RNAs targeting within a red blood cell antigen target locus; (b) adding a repair template comprising a mutation in the target locus flanked by a homology arm on each side, wherein the template may additionally include a diagnostic restriction enzyme site at the target locus; (c) ligating the guide sequence of step (b) into a plasmid which also expresses a nuclease and, optionally, a selectable marker or a reporter gene; (d) transfecting pluripotent cells with the plasmid of step (c) in the presence of an HDR repair oligo; (e) cloning and testing the resulting reporter positive clones for expression of the antigen target of interest; and (f) culturing the resulting cells to expand their numbers or to create a differentiated cell type of interest.

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