公开(公告)号：US20200332259A1

公开(公告)日：2020-10-22

申请号：US16913741

申请日：2020-06-26

Applicant: VERSITI BLOOD RESEARCH INSTITUTE FOUNDATION, INC.

Inventor： PETER JAY NEWMAN ,  Sridhar Rao ,  Nanyan Zhang ,  Huiying Zhi

IPC: C12N5/0789 ,  C12N9/22 ,  C12N15/113 ,  C12N5/078

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.