Removable plant transgenic loci with cognate guide rna recognition sites

Abstract

Transgenic plants comprising an originator guide RNA recognition site (OgRRS) as well as an exogenous cognate guide RNA recognition site (CgRRS) which is introduced at or near the junctions of the transgene insert, methods of making such plants, and use of such plants to facilitate breeding are disclosed.

What is claimed is: 
     
         1 . An edited transgenic plant genome comprising a first modified transgenic locus that comprises:
 (i) a first originator guide RNA recognition site (OgRRS) comprising a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site, wherein the OgRRS is located in transgenic DNA, in non-transgenic plant genomic DNA, or in a combination thereof in a first DNA junction polynucleotide of the first modified transgenic locus; and   (ii) a first cognate guide RNA recognition site (CgRRS) comprising a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site located in a second DNA junction polynucleotide of the first modified transgenic locus, wherein the CgRRS is absent from transgenic plant genomes comprising a first original transgenic locus that is unmodified and wherein the OgRRS and the CgRRS can hybridize to one first guide RNA (gRNA).   
     
     
         2 . The edited transgenic plant genome of  claim 1 , wherein the guide RNA hybridization site of the OgRRS and the CgRRS comprise at least 18 nucleotides of identical DNA sequence. 
     
     
         3 . The edited transgenic plant genome of  claim 1 , wherein the OgRRS and the CgRRS can be cleaved by the same RNA dependent DNA endonuclease (RdDe) when the OgRRS and the CgRRS are hybridized to the one first gRNA. 
     
     
         4 . The edited transgenic plant genome of  claim 3 , wherein the RdDe is a class 2 type II or class 2 type V RdDe. 
     
     
         5 . The edited transgenic plant genome of  claim 1 , wherein the PAM site in the OgRRS and the PAM site in the CgRRS comprise distinct PAM sequences that are recognized by the same RdDe when the OgRRS and the CgRRS are hybridized to the one gRNA. 
     
     
         6 . The edited transgenic plant genome of  claim 1 , wherein the OgRRS is located in non-transgenic DNA or in non-transgenic DNA and within about 1000, 750, 500, 250, 100, or 50 base pairs (bp) of transgenic DNA of the first DNA junction polynucleotide and/or wherein the CgRRS is located in non-transgenic DNA or in non-transgenic DNA and within about 1000, 750, 500, 250, 100, or 50 bp of transgenic DNA in the second DNA junction polynucleotide. 
     
     
         7 . The edited transgenic plant genome of  claim 1 , wherein the edited transgenic plant genome further comprises a second modified transgenic locus that comprises:
 (i) a second OgRRS located in transgenic DNA, non-transgenic plant genomic DNA, or in a combination thereof in a first DNA junction polynucleotide of the second modified transgenic locus; and   (ii) a second CgRRS located in a second DNA junction polynucleotide of the second modified transgenic locus, wherein the CgRRS is absent from unedited transgenic plant genomes comprising a second original transgenic locus, optionally wherein the second OgRRS and the second CgRRS can hybridize to one second guide RNA (gRNA) and optionally wherein the second guide RNA is not complementary to the first OgRRS and the first CgRRS.   
     
     
         8 . The edited transgenic plant genome of  claim 1 , wherein the first modified transgenic locus comprises at least one modification of a Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, 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, or TC1507 original transgenic locus in a transgenic corn plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus, and optionally wherein the modification further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus. 
     
     
         9 . The edited transgenic plant genome of  claim 1 , wherein the first modified transgenic locus comprises a modification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 original transgenic locus in a transgenic soybean plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus. 
     
     
         10 . The edited transgenic plant genome of  claim 1 , wherein the first modified transgenic locus comprises at least one modification of a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, or MON88913 original transgenic locus in a transgenic cotton plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the second modified transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus. 
     
     
         11 . The edited transgenic plant genome of  claim 1 , wherein the first modified transgenic locus comprises a modification of an GT73, HCN28, MON88302, or MS8 original transgenic locus in a transgenic canola plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus. 
     
     
         12 . The edited transgenic plant genome of any one of claims to  1  to  11 , wherein the first and/or second modified transgenic locus lacks a selectable marker transgene which confers resistance to an antibiotic, tolerance to an herbicide, or an ability to grow on a specific carbon source, optionally wherein the selectable marker transgene was present in the original transgenic locus and/or wherein the specific carbon source is optionally mannose. 
     
     
         13 . The edited transgenic plant genome of any one of  claims 1  to  11 , wherein: (i) the first CgRRS is located in non-transgenic plant genomic DNA of the second DNA junction polynucleotide of the first modified transgenic locus; or (ii) the first CgRRS is located in transgenic DNA of the second DNA junction polynucleotide of the first modified transgenic locus. 
     
     
         14 . The edited transgenic plant genome of  claim 13 , wherein the CgRRS is located in the non-transgenic plant genomic DNA and comprises a sequence having 50% to 70% sequence identity to the non-transgenic plant genomic DNA located at the same chromosomal location in the original transgenic locus. 
     
     
         15 . The edited transgenic plant genome of  claims 1  to  11 , wherein the first and/or second transgenic locus further comprise a second introduced transgene or optionally wherein the second introduced transgene is integrated at a site in the modified transgenic locus which was occupied by a selectable marker transgene in the original transgenic locus. 
     
     
         16 . The edited transgenic plant genome of any one of  claims 1  to  11 , wherein the genome further comprises a targeted genetic change. 
     
     
         17 . A transgenic plant cell comprising the edited transgenic plant genome of any one of  claims 1  to  11 . 
     
     
         18 . A transgenic plant comprising the transgenic plant genome of any one of  claims 1  to  11 . 
     
     
         19 . A transgenic plant part comprising the edited transgenic plant genome of any one of  claims 1  to  11 . 
     
     
         20 . The transgenic plant part of  claim 19 , wherein the part is a seed, leaf, tuber, stem, root, or boll. 
     
     
         21 . A method for obtaining a bulked population of inbred seed for commercial seed production comprising selfing the transgenic plant of  claim 18  and harvesting seed from the selfed crop plants. 
     
     
         22 . A method of obtaining hybrid crop seed comprising crossing a first crop plant comprising the transgenic plant of  claim 18  to a second crop plant and harvesting seed from the cross. 
     
     
         23 . The method of  claim 22 , wherein the first crop plant and the second crop plant are in distinct heterotic groups. 
     
     
         24 . The method of  claim 22 , wherein either the first or second crop plant are pollen recipients which have been rendered male sterile. 
     
     
         25 . The method of  claim 24 , wherein the crop plant is rendered male sterile by emasculation, cytoplasmic male sterility, a chemical hybridizing agent or system, a transgene, and/or a mutation in an endogenous plant gene. 
     
     
         26 . The method of any one of  claims 22  to  25 , further comprising the step of sowing the hybrid crop seed. 
     
     
         27 . A DNA molecule comprising the cognate guide RNA recognition site (CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNA comprises DNA sequences of the second DNA junction polynucleotide of the first modified transgenic locus set forth in any one of  claims 1  to  11 . 
     
     
         28 . The DNA of  claim 27 , wherein the modified transgenic locus is a Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, 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, or TC1507 transgenic locus. 
     
     
         29 . The DNA of  claim 27 , wherein the modified transgenic locus is an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, and/or SYHT0H2 transgenic locus. 
     
     
         30 . The DNA of  claim 27  wherein the modified transgenic locus is: (i) a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus and wherein the modifications optionally further comprise a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus; or (ii) wherein the modified transgenic locus is a GT73, HCN28, MON88302, or MS8 transgenic locus. 
     
     
         31 . The DNA of any one of  claims 27  to  30 , wherein the DNA is purified or isolated. 
     
     
         32 . A processed transgenic plant product containing the DNA of any one of  claims 27  to  30 . 
     
     
         33 . A biological sample containing the DNA of any one of  claims 27  to  30 . 
     
     
         34 . A nucleic acid marker adapted for detection of genomic DNA or fragments thereof comprising a cognate guide RNA recognition site (CgRRS) in, adjacent to, or operably linked to a DNA junction polynucleotide of a modified transgenic locus. 
     
     
         35 . The nucleic acid marker of  claim 34 , comprising a polynucleotide of at least 18 nucleotides in length which spans DNA sequences comprising both the CgRRS and non-transgenic plant genomic DNA flanking either a telomere-proximal and a centromere proximal end of the CgRRS. 
     
     
         36 . The nucleic acid marker of  claim 34 , wherein the marker further comprises a detectable label. 
     
     
         37 . The nucleic acid marker of  claim 34 , wherein the CgRRS and non-transgenic plant genomic DNA are identical to sequences in a modified transgenic locus. 
     
     
         38 . The nucleic acid marker of  claim 34 , wherein the modified transgenic locus is a Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MIR162, 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, or TC1507 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus. 
     
     
         39 . The nucleic acid marker of  claim 34 , wherein the modified transgenic locus is a modified A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus. 
     
     
         40 . The nucleic acid marker of  claim 34 , wherein the modified transgenic locus is a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus. 
     
     
         41 . The nucleic acid marker of  claim 34 , wherein the modified transgenic locus is a GT73, HCN28, MON88302, or MS8 transgenic locus comprising the CgRRS, wherein the CgRRS is located in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus. 
     
     
         42 . A processed transgenic plant product obtained from the transgenic plant part of  claim 19  or  20 , wherein the processed plant product contains a polynucleotide comprising a cognate guide RNA recognition site (CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNA comprises transgenic or non-transgenic plant genomic DNA sequences of the second DNA junction polynucleotide of the first modified transgenic locus. 
     
     
         43 . A biological sample obtained from the transgenic plant cell of  claim 17 , the transgenic plant of  claim 18 , or the transgenic plant part of  claim 19  or  20 , wherein the biological sample contains one or more polynucleotide(s) comprising the a cognate guide RNA recognition site (CgRRS) and at least 10 bp of transgenic DNA or non-transgenic plant genomic DNA flanking the CgRRS, wherein the transgenic DNA or non-transgenic plant genomic DNA comprises transgenic or non-transgenic plant genomic DNA sequences of the second DNA junction polynucleotide of the first modified transgenic locus. 
     
     
         44 . A method of detecting the edited transgenic plant genome of any one of  claims 1  to  11 , comprising the step of detecting the presence of a polynucleotide comprising one or more of said CgRRS. 
     
     
         45 . The method of  claim 44 , wherein the polynucleotide is detected by detecting a single nucleotide polymorphism (SNP) in the CgRRS that is present in the modified transgenic locus but absent in the original transgenic locus. 
     
     
         46 . The method of  claim 44 , wherein the edited transgenic plant genome is detected in a transgenic plant cell, a transgenic plant part, a transgenic plant, a processed transgenic plant product, or a biological sample. 
     
     
         47 . A method of obtaining an edited transgenic plant genome comprising a modified transgenic locus comprising the step of introducing a cognate guide RNA recognition site (CgRRS) in a DNA junction polynucleotide of an original transgenic locus, wherein the CgRRS is in, adjacent to, or operably linked to a DNA junction polynucleotide of the modified transgenic locus. 
     
     
         48 . The method of  claim 47 , wherein the CgRRS is located in non-transgenic plant genomic DNA of the DNA junction polynucleotide of the modified transgenic locus and optionally wherein the CgRRS comprises a sequence having 50% to 70% sequence identity to the non-transgenic plant genomic DNA located at the same chromosomal location in the original transgenic locus. 
     
     
         49 . The method of  claim 47 , wherein the CgRRS is introduced by:
 (a) contacting the original transgenic locus with: (i) an RNA dependent DNA endonuclease (RdDe) or RdDe nickase; and a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within the DNA junction polynucleotide of the original transgenic locus; or (ii) a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within the DNA junction polynucleotide of the original transgenic locus; and (iii) a donor DNA template spanning a double stranded DNA break site in the DNA junction polynucleotide, wherein the donor DNA template comprises a guide RNA hybridization site of the CgRRS, optionally a PAM site of the CgRRS, and optionally DNA sequences of the DNA junction polynucleotide flanking the double stranded DNA break site; and   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the CgRRS.   
     
     
         50 . The method of  claim 47 , wherein the CgRRS is introduced by:
 (a) contacting the original transgenic locus with: (i) at least one adenine base editor (ABE) and/or cytosine base pair editor (CBE); and (ii) a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within the DNA junction polynucleotide of the original transgenic locus; and   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the CgRRS.   
     
     
         51 . The method of  claim 47 , wherein the CgRRS is introduced by:
 (a) contacting the original transgenic locus with: (i) a Zinc Finger Nuclease or TALEN which recognizes a DNA junction polynucleotide of the original transgenic locus or (ii) a Zinc Finger nickase or Tale nickase which recognizes a DNA junction polynucleotide of the original transgenic locus; and (iii) a donor DNA template spanning a double stranded DNA break site in the DNA junction polynucleotide, wherein the donor DNA template comprises a guide RNA hybridization site of the CgRRS, optionally a PAM site of the CgRRS, and optionally DNA sequences of the DNA junction polynucleotide flanking the double stranded DNA break site; and   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the CgRRS.   
     
     
         52 . The method of  claim 47 , further comprising contacting the original transgenic locus with one or more gene editing molecules that provide for excision or inactivation of a selectable marker transgene of the original transgenic locus and selecting for a transgenic plant cell, transgenic plant part, or transgenic plant wherein the selectable marker transgene has been excised or inactivated. 
     
     
         53 . The method of  claim 52 , wherein the gene editing molecules include a donor DNA template or other DNA template containing an expression cassette or coding region which confers a useful trait and the transgenic plant cell, transgenic plant part, or transgenic plant is selected for integration of the expression cassette at the site of the selectable marker transgene excision or inactivation. 
     
     
         54 . A method of excising a modified transgenic locus or portion thereof from an edited transgenic plant genome comprising the steps of:
 (a) contacting the edited transgenic plant genome of any one of  claims 1  to  11  with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the first OgRRS and the first CgRRS; wherein the RdDe recognizes a OgRRS/gRNA and a CgRRS/gRNA hybridization complex; and,   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the modified transgenic locus or portion thereof flanked by the first OgRRS and the first CgRRS has been excised.   
     
     
         55 . The method of  claim 54 , wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising or encoding the RdDe(s) and the gRNA into a transgenic plant cell comprising the edited transgenic plant genome. 
     
     
         56 . The method of  claim 54 , wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising the gRNA into a transgenic plant cell comprising the edited transgenic plant genome, wherein the transgenic plant cell comprises a polynucleotide that encodes the RdDe and the encoded RdDe is expressed in the transgenic plant cell. 
     
     
         57 . The method of  claim 54 , wherein the edited transgenic plant genome further comprises a second modified transgenic locus that comprises a second OgRRS located in non-transgenic plant genomic DNA of a first DNA junction polynucleotide of the second modified transgenic locus; and a second CgRRS located in non-transgenic plant genomic DNA of a second DNA junction polynucleotide of the second modified transgenic locus; and
 wherein the method further comprises:
 (a) contacting the edited transgenic plant genome with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the second OgRRS and the second CgRRS; wherein the RdDe recognizes a second OgRRS/gRNA and a second CgRRS/gRNA hybridization complex; and, 
 (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the second modified transgenic locus or portion thereof flanked by the second OgRRS and the second CgRRS has been excised. 
   
     
     
         58 . The method of  claim 54 , wherein the transgenic plant cell is in tissue culture, in a callus culture, a plant part, or in a whole plant and/or wherein the transgenic plant cell is a haploid plant cell. 
     
     
         59 . The method of  claim 54 , wherein the edited transgenic plant genome is further contacted in step (a) with a donor DNA template, optionally wherein the donor DNA template comprises a polynucleotide sequence comprising non-transgenic plant genomic DNA located outside of the modified transgenic DNA which is to be excised, optionally wherein the donor DNA template comprises non-transgenic plant genomic DNA present in an unmodified plant at the site where the transgenic locus was originally inserted, and optionally in step (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein polynucleotides in any of the donor DNA templates have integrated in the position in the plant genome where the modified transgenic locus was excised. 
     
     
         60 . A method of obtaining a plant breeding line comprising:
 (a) crossing two transgenic plants comprising the edited transgenic genomes of any of  claims 1  to  11 , wherein a first plant comprising the first modified transgenic locus is crossed to a second plant comprising the second modified transgenic locus; and,   (b) selecting a progeny plant comprising the first and second modified transgenic locus from the cross, thereby obtaining a plant breeding line.   
     
     
         61 . The method of  claim 60 , wherein the plant breeding line is subjected to a haploid inducer and a haploid plant breeding line comprising at least the first and second breeding line is selected. 
     
     
         62 . A method for obtaining inbred transgenic plant germplasm containing different transgenic traits comprising:
 (a) introgressing at least a first transgenic locus and a second transgenic locus into inbred germplasm to obtain a donor inbred parent plant line comprising the first and second transgenic loci, wherein a first OgRRS and a first CgRRS are operably linked to both DNA junction polynucleotides of at least the first transgenic locus and optionally wherein a second OgRRS and a second CgRRS are operably linked to the second transgenic locus;   (b) contacting the transgenic plant genome of the donor inbred parent plant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the first OgRRS and the second CgRRS; wherein the RdDe recognizes a first OgRRS/gRNA and a first CgRRS/gRNA hybridization complex; and   (c) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising an edited transgenic plant genome in the inbred germplasm, wherein the first transgenic locus has been excised and the second transgenic locus is present in the inbred germplasm.   
     
     
         63 . The method of  claim 62 , wherein the introgression comprises crossing germplasm comprising the first and/or second transgenic plant locus with the inbred germplasm, selecting progeny comprising the first or second transgenic plant locus, and crossing the selected progeny with the inbred germplasm as a recurrent parent. 
     
     
         64 . The method of  claim 62 , further comprising contacting the transgenic plant genome in step (b) with one or more gene editing molecules that provide for excision or inactivation of a selectable marker transgene of the second transgenic locus and selecting for a transgenic plant cell, transgenic plant part, or transgenic plant wherein the selectable marker transgene has been excised or inactivated. 
     
     
         65 . The method of  claim 62 , wherein the gene editing molecules include a donor DNA template containing an expression cassette or coding region which confers a useful trait and the transgenic plant cell, transgenic plant part, or transgenic plant is selected for integration of the expression cassette at the site of the selectable marker transgene excision or inactivation. 
     
     
         66 . The method of  claim 62 , wherein a second OgRRS and a second CgRRS are operably linked to the second transgenic locus and wherein the method further comprises contacting the transgenic plant genome of the donor inbred parent plant line with: (i) an RNA dependent DNA endonuclease (RdDe); and (ii) a guide RNA (gRNA) capable of hybridizing to the guide RNA hybridization site of the second OgRRS and the second CgRRS; wherein the RdDe recognizes a second OgRRS/gRNA and a second CgRRS/gRNA hybridization complex in step (b); and selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the second transgenic locus has been excised in step (c). 
     
     
         67 . The method of  claim 62 , wherein the transgenic plant genome is contacted in step (b) by introducing one or more compositions comprising or encoding the RdDe(s) and gRNAs into a transgenic plant cell comprising the transgenic plant genome. 
     
     
         68 . The method of  claim 62 , wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising the gRNA into a transgenic plant cell comprising the edited transgenic plant genome, wherein the transgenic plant cell comprises a polynucleotide that encodes the RdDe and the encoded RdDe is expressed in the transgenic plant cell. 
     
     
         69 . The method of  claim 62 , wherein the transgenic plant genome is further contacted in step (b) with a donor DNA template molecule comprising an introduced transgene and a transgenic plant cell comprising an edited transgenic plant genome comprising an insertion of the introduced transgene in the first transgenic locus is selected in step (c). 
     
     
         70 . The method of  claim 62 , wherein the transgenic plant genome is further contacted in step (b) with: (i) a donor DNA template molecule comprising an introduced transgene; and (ii) one or more DNA editing molecules which introduce a double stranded DNA break in the second transgenic locus; and a transgenic plant cell comprising an edited transgenic plant genome comprising an insertion of the introduced transgene in the second transgenic locus is selected in step (b). 
     
     
         71 . The method of  claim 62 , further comprising:
 (d) contacting the edited transgenic plant genome in the selected transgenic plant cell of step (c) with: (i) a donor DNA template molecule comprising an introduced transgene; and (ii) one or more DNA editing molecules which introduce a double stranded DNA break in or near the excision site of the first transgenic locus or in the second transgenic locus; and,   (e) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising a further edited transgenic plant genome comprising an insertion of the introduced transgene in or near the excision site of the first transgenic locus or in the second transgenic locus.   
     
     
         72 . The method of any one of  claims 62  to  71 , wherein the transgenic plant germplasm is transgenic corn plant germplasm and wherein the first and/or second modified transgenic locus comprises at least one modification of a Bt11, DAS-59122-7, DP-4114, GA21, MON810, MON87411, MON87427, MON88017, MON89034, MIR162, 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, or TC1507 original transgenic locus in a transgenic corn plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus. 
     
     
         73 . The method of any one of  claims 62  to  71 , wherein the transgenic plant germplasm is transgenic soybean plant germplasm and wherein the first and/or second modified transgenic locus comprises a modification of an A5547-127, DAS44406-6, DAS68416-4, DAS81419-2, GTS 40-3-2, MON87701, MON87708, MON89788, MST-FGØ72-3, or SYHT0H2 original transgenic locus in a transgenic soybean plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus. 
     
     
         74 . The method of any one of  claims 62  to  71 , wherein the transgenic plant germplasm is transgenic cotton plant germplasm and wherein the first, second, and/or third modified transgenic locus comprises at least one modification of a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, or MON88913 original transgenic locus in a transgenic cotton plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus. 
     
     
         75 . The method of any one of  claims 62  to  71 , wherein the transgenic plant germplasm is transgenic canola plant germplasm and wherein the first, second, and or third modified transgenic locus comprises a modification of an GT73, HCN28, MON88302, or MS8 original transgenic locus in a transgenic canola plant genome, wherein the modification comprises the CgRRS in the second DNA junction polynucleotide of the first transgenic locus and optionally the CgRRS in the second DNA junction polynucleotide of the transgenic locus, and wherein the modification optionally further comprises a deletion of at least one selectable marker gene and/or non-essential DNA in the original transgenic locus.