Generation of plants with improved transgenic loci by genome editing

Abstract

Methods of selectively excising polynucleotide segments including segments comprising non-essential DNA and/or selectable marker genes from transgenic loci from transgenic plants as well as making targeted genetic changes with genome editing techniques are provided. Also provided are transgenic plants comprising the transgenic loci lacking the non-essential DNA and/or selectable markers and use of such methods and plants to facilitate plant breeding are disclosed.

1 - 33 . (canceled) 
     
     
         34 . A method of modifying a transgenic event in a plant by deleting at least one copy of a repetitive sequence in the transgenic event with genome editing molecules, wherein the repetitive sequence comprises a fragment of a transgene, a fragment of a transgene element, and/or an  Agrobacterium  right and/or left border sequence. 
     
     
         35 . The method of  claim 34 , wherein:
 (a) the approved transgenic locus is DP-4114;   (b) the approved transgenic locus is 1507, optionally wherein the repetitive sequence comprises a truncated cry1F fragment of 335 bp located at the 5′ end of the insertion locus and/or at least one of incomplete sequences from the pat gene, the maize ubiquitin promoter, the mannopine synthase terminator from  Agrobacterium , fragments of chloroplast DNA, and sequences with similarity to retrotransposons present in the border region of the insert;   (c) the approved transgenic locus is MIR604; or   (d) the approved transgenic locus is DAS44406-6.   
     
     
         36 . The method of  claim 34 , wherein the use of genome editing molecules comprises:
 (a) contacting a transgenic plant genome with one or more gene editing molecules which introduce one or more single or double-stranded breaks providing for excision of a segment of the original transgenic locus comprising, consisting essentially of, or consisting of the fragment of a transgene, the fragment of a transgene element, and/or the  Agrobacterium  right and/or left border sequence,   wherein the transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising or encoding the gene editing molecules into a plant cell comprising the transgenic plant genome.   
     
     
         37 . The method of  claim 36 , further comprising:
 (b) selecting a plant cell, plant part, or plant containing a modified transgenic locus, wherein a segment comprising, consisting essentially of, or consisting of the fragment of a transgene, the fragment of a transgene element, and/or the  Agrobacterium  right and/or left border sequence has been deleted,   thereby obtaining a plant cell, plant part, or plant containing the modified transgenic event.   
     
     
         38 . The method of  claim 34 , wherein a selectable marker gene is also removed with genome editing molecules. 
     
     
         39 . The method of  claim 38 , further comprising contacting the genome with one or more gene editing molecules which introduce one or more single or double-stranded breaks providing for excision of a segment comprising, consisting essentially of, or consisting of the selectable marker gene;
 and in step (b) selecting a plant cell, plant part, or plant containing a modified transgenic locus, wherein a selectable marker gene and the segment comprising, consisting essentially of, or consisting of the fragment of a transgene, the fragment of a transgene element, and/or the  Agrobacterium  right and/or left border sequence within the transgenic event have been deleted.   
     
     
         40 . The method of  claim 34 , wherein the segment comprising, the fragment of a transgene, the fragment of a transgene element, and/or the  Agrobacterium  right and/or left border sequence is also the segment comprising, consisting essentially of, or consisting of the selectable marker gene. 
     
     
         41 . The method of  claim 34 , the fragment of a transgene, the fragment of a transgene element, and/or the  Agrobacterium  right and/or left border sequence is a different segment from the segment comprising, consisting essentially of, or consisting of the selectable marker gene. 
     
     
         42 . The method of  claim 34 , wherein the one or more gene editing molecules is selected from the group consisting of RNA dependent DNA endonucleases (RdDe) and guide RNAs, RNA dependent nickases and guide RNAs, Zinc Finger nucleases or nickases, and TALE nucleases or nickases. 
     
     
         43 - 49 . (canceled) 
     
     
         50 . The method of  claim 34 , wherein the modification comprises a modification of:
 (i) a Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR604, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, 98140, and/or TC1507 transgenic locus in a transgenic maize plant genome; or   (ii) an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, and/or SYHTOH2 transgenic locus in a transgenic soybean plant genome.   
     
     
         51 . A transgenic plant comprising a modified transgenic event comprising a deletion of at least one copy of a repetitive sequence in the transgenic event comprising a fragment of a transgene, a fragment of a transgene element, and/or an  Agrobacterium  right and/or left border sequence. 
     
     
         52 . The transgenic plant of  claim 51 , wherein:
 (a) the DP-4114 transgenic event is modified;   (b) the 1507 transgenic event is modified, and wherein the repetitive sequence comprises a truncated cry1F fragment of 335 bp located at the 5′ end of the insertion locus and/or at least one of incomplete sequences from the pat gene, the maize ubiquitin promoter, the mannopine synthase terminator from  Agrobacterium , fragments of chloroplast DNA, and sequences with similarity to retrotransposons present in the border region of the insert;   (c) the MIR604 transgenic event is modified; or   (d) the DAS44406-6 transgenic event is modified.   
     
     
         53 . (canceled) 
     
     
         54 . The transgenic plant of  claim 51 , wherein a selectable marker gene is also removed with genome editing molecules. 
     
     
         55 . (canceled) 
     
     
         56 . The transgenic plant of  claim 51 , wherein the repetitive sequence to be deleted is flanked by operably linked protospacer adjacent motif (PAM) sites in the unmodified transgenic event and/or wherein the repetitive sequence to be deleted encompasses an operably linked PAM site in the unmodified transgenic event. 
     
     
         57 . The transgenic plant of  claim 51 , wherein the modified transgenic event comprises PAM sites flanking the excision site of the deleted repetitive sequence. 
     
     
         58 . The transgenic plant of  claim 56 , wherein the PAM sites are recognized by an RNA dependent DNA endonuclease (RdDe); optionally wherein the RdDe is a class 2 type II or class 2 type V RdDe. 
     
     
         59 . The transgenic plant of  claim 51 , wherein the modified transgenic event comprises two or more deletions. 
     
     
         60 - 61 . (canceled) 
     
     
         62 . The transgenic plant of  claim 51 , wherein the modification comprises a modification of:
 (i) a Bt11, DAS-59122-7, GA21, MON810, MON87411, MON87427, MON88017, MON89034, NK603, SYN-E3272-5, 5307, DAS-40278, DP-32138, DP-33121, HCEM485, LY038, MON863, MON87403, MON87403, MON87419, MON87460, MZHG0JG, MZIR098, VCO-Ø1981-5, and/or 98140, transgenic event in a transgenic maize plant genome;   (ii) an A5547-127, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, and/or SYHTOH2 transgenic locus in a transgenic soybean plant genome.   
     
     
         63 . A transgenic plant seed obtained from the plant of  claim 51 , wherein the seed comprises the modified transgenic event. 
     
     
         64 . A transgenic plant cell obtained from the plant of  claim 51 , wherein the plant cell comprises the modified transgenic event. 
     
     
         65 - 95 . (canceled)