Targeted Integration And Stacking Of DNA Through Homologous Recombination

Abstract

The invention provides various methods for the targeted integration and stacking of nucleotide sequences in the genome of a host plant cell using homologous recombination.

1 - 14 . (canceled) 
     
     
         15 . A method of preparing a host cell having a genome with a target site for reiterative gene stacking in the host cell genome, the method comprising the steps of:
 (a) providing a host cell having a genome comprising a target sequence comprising:
 (i) a truncated functional sequence; and 
 (ii) a host homology sequence; 
   (b) introducing into the host cell a donor sequence comprising:
 (i) a heterologous sequence of interest or fragment thereof; 
 (ii) a donor homology sequence homologous to the host homology sequence; 
 (iii) a sequence that completes the truncated functional sequence located between the heterologous sequence of interest or fragment thereof and the donor homology sequence; 
 (iv) a first recombinase recognition site located between the donor homology sequence and the sequence that completes the truncated functional sequence; and 
 (v) a second recombinase recognition site located between the sequence that completes the truncated functional sequence and the heterologous sequence of interest or fragment thereof; and further wherein the first and second recombinase recognition sites are oriented relative to one another such that the sequence that completes the truncated functional sequence is excisable in the presence of a recombinase; 
   (c) obtaining in the host cell a recombination product comprising:
 (i) the heterologous sequence of interest; 
 (ii) a recombined sequence resulting from homologous recombination of the host homology sequence and the donor homology sequence; 
 (iii) a restored functional sequence comprising the truncated functional sequence, the sequence that completes the truncated functional sequence, the recombined sequence located between the truncated functional sequence and the sequence that completes the truncated functional sequence, and the first recombinase recognition site located between the sequence that completes the truncated functional sequence and the recombined sequence; and 
 (iv) the second recombinase recognition site located between the heterologous sequence of interest and the restored functional sequence. 
   
     
     
         16 . The method of  claim 15 , wherein the restored functional sequence encodes a marker. 
     
     
         17 . The method of  claim 16 , wherein the marker is selected from the group consisting of NPTII, HPT, PAT, BAR, EPSPS, GAT, HPPD, ALS, PPO, PMI, GUS, LUC, GFP, RFP, and CFP. 
     
     
         18 . A host cell produced by the method of  claim 15 . 
     
     
         19 . The host cell of  claim 18 , which is a plant cell. 
     
     
         20 . A plant or plant part comprising the plant cell of  claim 19 . 
     
     
         21 . The method of  claim 15 , wherein the target sequence of step (a) further comprises (iii) a second host homology sequence, and wherein the host homology sequence of (a)(ii) is located between the second host homology sequence and the truncated functional sequence; and
 wherein the donor sequence of step (b) further comprises (vi) a second donor homology sequence having homology to the second host homology sequence, and wherein the heterologous sequence of interest is located between the second donor homology sequence and the second recombinase recognition site; and   wherein the recombination product of step (c) further comprises (v) a second recombined sequence resulting from homologous recombination of the second host homology sequence and the second donor homology sequence, and wherein the heterologous sequence of interest of step (c)(1) is located between the second recombined sequence and the second recombinase recognition site.   
     
     
         22 . A host cell produced by the method of  claim 21 . 
     
     
         23 . The host cell of  claim 22 , which is a plant cell. 
     
     
         24 . A plant or plant part comprising the plant cell of  claim 23 . 
     
     
         25 . The method of  claim 15 , further comprising:
 (d) introducing into the host cell of step (c) a recombinase or recombinase coding sequence to thereby excise the sequence that completes the truncated functional sequence and yield an excision product comprising:
 (i) the heterologous sequence of interest; 
 (ii) the truncated functional sequence; 
 (iii) the recombined sequence located between the heterologous sequence of interest and the truncated functional sequence; and 
 (iv) a regenerated target recombinase recognition site located between the heterologous sequence of interest and the recombined sequence.