Nucleic acid constructs comprising gene editing multi-sites and uses thereof

Abstract

Disclosed herein is a polynucleotide construct comprising one or more nuclease recognition sequences upstream and downstream of a Gene editing multi-site that comprises a plurality of nuclease recognition sequences. The plurality of nuclease recognition sequences facilitate insertion of one or more exogenous donor genes into the host cell.

1 . (canceled) 
     
     
         2 . A method for generating a genetically modified mammalian cell, said method comprising introducing an exogenous nucleic acid sequence that encodes an exogenous protein in a mammalian cell that comprises a GEMS sequence that is inserted within a genome of said mammalian cell,
 wherein said GEMS sequence is heterologous to said genome,   wherein said GEMS sequence comprises a plurality of nuclease recognition sequences that comprises a target sequence and a protospacer adjacent motif (PAM) sequence or reverse complements thereof,   and wherein said exogenous nucleic acid sequence is inserted within at least one of said plurality of nuclease recognition sequences, thereby generating a genetically modified mammalian cell.   
     
     
         3 . The method of  claim 2 , wherein said exogenous protein comprises an antibody, a chimeric antigen receptor (CAR), a T-cell receptor (TCR), a B-cell receptor (BCR), an αβ receptor, a γδ T-receptor, or a combination thereof. 
     
     
         4 . The method of  claim 2 , wherein said mammalian cell is a stem cell. 
     
     
         5 . The method of  claim 4 , wherein said stem cell is an embryonic stem cell, non-embryonic stem cell, pluripotent stem cell, placental stem cell, induced pluripotent stem cell, trophoblast stem cell, adult stem cell, or a hematopoietic stem cell. 
     
     
         6 . The method of  claim 2 , wherein said mammalian cell is a progenitor cell, a T cell, or a natural killer (NK) cell. 
     
     
         7 . The method of  claim 6 , wherein said T cell is a T-cell is an αβ T-cell, an NK T-cell, a γδ T-cell, a regulatory T-cell, a T helper cell and a cytotoxic T-cell. 
     
     
         8 . A genetically modified mammalian cell generated by the method of  claim 2 .