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.

Abstract: Methods of creating cells expressing specific platelet alloantigens by combining gene editing techniques and cell culture differentiation or expansion techniques employing pluripotent cells, including the steps of transfecting pluripotent cells with a plasmid encoding one or more guide RNAs targeting within a platelet alloantigen target locus and a nuclease in the presence of an HDR repair oligo and culturing the resulting cells to expand their numbers or to create a differentiated cell type of interest.

Abstract: A method of creating cells expressing specific platelet alloantigens is disclosed. In one embodiment, the method comprises the steps of (a) combining one or more guide RNAs targeting within a platelet alloantigen 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 alloantigen target of interest; and (f) culturing the resulting cells to expand their numbers or to create a differentiated cell type of interest.