Methods for genomic integration

Abstract

Provided herein are methods of integrating one or more exogenous nucleic acids into one or more selected target sites of a host cell genome. In certain embodiments, the methods comprise contacting the host cell genome with one or more integration polynucleotides comprising an exogenous nucleic acid to be integrated into a genomic target site, a nuclease capable of causing a break at the genomic target site, and a linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the population of cells, whereupon said homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker. In some embodiments, the methods further comprise selecting a host cell that expresses the selectable marker.

1 .- 146 . (canceled) 
     
     
         147 . A composition comprising:
 (a) a site-specific nuclease, or a nucleic acid comprising a coding sequence for a site-specific nuclease; and   (b) a linear nucleic acid comprising two internal homology regions that are capable of homologously recombining with each other in a host cell, whereupon homologous recombination of the internal homology regions results in formation of a circular nucleic acid comprising a coding sequence for a selectable marker.   
     
     
         148 . A host cell comprising the composition of  claim 147 . 
     
     
         149 . A cell culture composition comprising a cell culture medium and the host cell of any one of  claim 148 . 
     
     
         150 . The cell culture composition of  claim 149 , further comprising a compound that selects for expression of the selectable marker. 
     
     
         151 . A linear nucleic acid comprising a first homology region (HR1) and a second homology region (HR2), wherein HR1 and HR2 are capable of recombining with each other via homologous recombination, whereupon homologous recombination of HR1 with HR2 results in formation of a circular nucleic acid comprising a coding sequence for a selectable marker. 
     
     
         152 . A composition for integrating one or more exogenous nucleic acids into one or more target sites of a host cell genome, the composition comprising:
 (a) one or more exogenous donor nucleic acids (ES) capable of recombining, via homologous recombination, at one or more target sites (TS) of a host cell genome;   (b) an RNA-guided endonuclease (RGEN), or a nucleic acid encoding said RGEN;   (c) one or more ribonucleic acids that enable site-specific recognition and cleavage of the one or more TS by the RGEN, or one or more nucleic acids encoding said one or more ribonucleic acids; and   (d) a linear pre-recombination nucleic acid capable of in vivo homologous recombination with itself or with one or more additional linear pre-recombination nucleic acids in the composition, whereupon said in vivo homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker.   
     
     
         153 . A host cell comprising the composition of  claim 152 .