Methods of genome editing of cells with modified donor templates

Abstract

Described herein is a DNA template plasmid for generating genome modified immune cells including a plasmid backbone, a first insert, and a second insert. The first insert includes a transgene, wherein the transgene is flanked by left and right homology arms that are complementary to sequences on both sides of a cleavage site in a target expressed gene in an unmodified immune cell. The second insert includes a cleavage target including a protospacer sequence defining the cleavage site in the target expressed gene, and a protoadjacent motif sequence (PAM) for recognition by a Cas9 ribonucleoprotein complex (Cas9-RNP). The Cas9-RNP includes a Cas9 polypeptide and a single guide RNA (sgRNA) comprising a sequence complementary to the protospacer sequence. The Cas9-RNP binds the second insert and linearizes the DNA template plasmid by Cas9-RNP-directed cleavage at the cleavage site. Also included are RNP complexes, methods of genome modifying immune cells, and treatment methods.

Dependencies are inferred from the claim text.