Excisable plant transgenic loci with signature protospacer adjacent motifs or signature guide rna recognition sites

Abstract

Transgenic plants comprising synthetic protospacer adjacent motifs (sPAMs) or synthetic guide RNA recognition sites introduced at or near the junctions the transgene insert with non-transgenic genomic DNA, 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 set of signature protospacer adjacent motif (sPAM) sites and/or signature guide RNA recognition (sigRNAR) sites, wherein the sPAM and/or sigRNAR sites are operably linked to both DNA junction polynucleotides of a first modified transgenic locus in the transgenic plant genome and wherein the sPAM and/or sigRNAR sites are absent from a transgenic plant genome comprising an original transgenic locus. 
     
     
         2 . An edited transgenic plant genome comprising a signature protospacer adjacent motif (sPAM) site and/or signature guide RNA recognition (sigRNAR) site, wherein the sPAM and/or sigRNAR site is operably linked to a DNA junction polynucleotides of a first modified transgenic locus in the transgenic plant genome and wherein the sPAM and/or sigRNAR site is absent from a transgenic plant genome comprising an original transgenic locus. 
     
     
         3 . The edited transgenic plant genome of  claim 1 , wherein the first set of sPAM and/or sigRNAR sites are recognized by the same RNA dependent DNA endonuclease (RdDe) or same class of RdDe. 
     
     
         4 . The edited transgenic plant genome of  claim 1 , wherein the first set of sigRNAR sites are recognized by the same RNA dependent DNA endonuclease (RdDe) or same class of RdDe and a first guide RNA. 
     
     
         5 . The edited transgenic plant genome of  claim 1 , wherein the genome further comprises a second set of sPAM and/or sigRNAR sites which are operably linked to both DNA junction polynucleotides of a second modified transgenic locus in the edited transgenic plant genome and wherein the second set of sPAM and/or sigRNAR sites are recognized by the same RdDe or same class of RdDe. 
     
     
         6 . The edited transgenic plant genome of  claim 1 , wherein (i) the first set of sPAM and/or sigRNAR sites and second set of sPAM and/or sigRNAR sites are each recognized by distinct RdDe or by distinct classes of RdDe. 
     
     
         7 . The edited transgenic plant genome of  claim 1 , wherein (i) the first set of sigRNAR sites and second set of sigRNAR sites are each respectively recognized by a first guide RNA and a guide RNA. 
     
     
         8 . The edited transgenic plant genome of  claim 1 , wherein the genome further comprises a third set of sPAM and/or sigRNAR sites which are operably linked to both DNA junction polynucleotides of a third modified transgenic locus in the edited transgenic plant genome and wherein the third set of sPAMs and/or sigRNAR are recognized by the same RdDe or same class of RdDe. 
     
     
         9 . The edited transgenic plant genome of  claim 8 , wherein the first, second, and third set of sigRNAR sites are each respectively recognized by a first guide RNA, a second guide RNA, and a third guide RNA. 
     
     
         10 . The edited transgenic plant genome of any one of  claims 1  to  9 , wherein the RdDe is a class 2 type II or class 2 type V RdDe. 
     
     
         11 . The edited transgenic plant genome of any one of  claims 1  to  9 , wherein the first, second, and/or third 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, wherein the specific carbon source is optionally mannose. 
     
     
         12 . The edited transgenic plant genome of  claim 11 , wherein the selectable marker transgene was present in the original transgenic locus. 
     
     
         13 . The edited transgenic plant genome of any one of  claims 1  to  9 , wherein the first, second, and/or third modified transgenic locus further comprise a second introduced transgene. 
     
     
         14 . The edited transgenic plant genome of  claim 1 , 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. 
     
     
         15 . The edited transgenic plant genome of any one of  claims 1  to  9 , wherein the first, second, and/or third 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 first, second, and/or third set of sPAM and/or sigRNAR sites in the DNA junction polynucleotides of the first, second, and/or third modified 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. 
     
     
         16 . The edited transgenic plant genome of any one of  claims 1  to  9 , wherein the first, second, and or third 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 first, second, and/or third set of sPAM and/or sigRNAR sites in the DNA junction polynucleotides of the first, second, and/or third modified 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. 
     
     
         17 . The edited transgenic plant genome of any one of  claims 1  to  9 , 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 first, second, and/or third set of sPAM and/or sigRNAR sites in the DNA junction polynucleotides of the first, second, and/or third modified 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. 
     
     
         18 . The edited transgenic plant genome of any one of  claims 1  to  9 , 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 first, second, and/or third set of sPAMs and/or sigRNAR sites in the DNA junction polynucleotides of the first, second, and/or third modified 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. 
     
     
         19 . The edited transgenic plant genome of any one of  claims 1  to  9 , wherein the genome further comprises a targeted genetic change. 
     
     
         20 . A transgenic plant cell comprising the edited transgenic plant genome of any one of  claims 1  to  9 . 
     
     
         21 . A transgenic plant comprising the transgenic plant genome of any one of  claims 1  to  9 . 
     
     
         22 . A transgenic plant part comprising the edited transgenic plant genome of any one of  claims 1  to  9 . 
     
     
         23 . The transgenic plant part of  claim 22 , wherein the part is a seed, leaf, tuber, stem, root, or boll. 
     
     
         24 . A method for obtaining a bulked population of inbred seed for commercial seed production comprising selfing the transgenic plant of  claim 21  and harvesting seed from the selfed elite crop plants. 
     
     
         25 . A method of obtaining hybrid crop seed comprising crossing a first crop plant comprising the transgenic plant of  claim 21 , to a second crop plant and harvesting seed from the cross. 
     
     
         26 . The method of  claim 25 , wherein the first crop plant and the second crop plant are in distinct heterotic groups. 
     
     
         27 . The method of  claim 25 , wherein either the first or second crop plant are pollen recipients which have been rendered male sterile. 
     
     
         28 . The method of  claim 27 , 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. 
     
     
         29 . The method of  claim 25 , further comprising the step of sowing the hybrid crop seed. 
     
     
         30 . DNA comprising a sPAM and/or sigRNAR in, adjacent to, or operably linked to one or both DNA junction polynucleotides of a modified transgenic locus. 
     
     
         31 . The DNA of  claim 30 , 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 and wherein the modifications optionally further comprise a deletion of at least one selectable marker gene and/or non-essential DNA in the transgenic locus. 
     
     
         32 . The DNA of  claim 30 , 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 and wherein the modifications optionally further comprise a deletion of at least one selectable marker gene and/or non-essential DNA in the transgenic locus. 
     
     
         33 . The DNA of  claim 30 , 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 and wherein the modifications optionally further comprise a deletion of at least one selectable marker gene and/or non-essential DNA in the transgenic locus. 
     
     
         34 . The DNA of any one of  claims 30  to  33 , wherein the DNA is purified or isolated. 
     
     
         35 . A processed transgenic plant product containing the DNA of any one of  claims 30  to  33 . 
     
     
         36 . A biological sample containing the DNA of any one of  claims 30  to  33 . 
     
     
         37 . A nucleic acid marker adapted for detection of genomic DNA or fragments thereof comprising a sPAM and/or sigRNAR in, adjacent to, or operably linked to one or both DNA junction polynucleotides of a modified transgenic locus. 
     
     
         38 . The nucleic acid marker of  claim 37 , comprising a polynucleotide of at least 18 nucleotides in length which spans the sPAM and/or sigRNAR. 
     
     
         39 . The nucleic acid marker of  claim 37 , wherein the marker further comprises a detectable label. 
     
     
         40 . The nucleic acid marker of  claim 37 , wherein the modified transgenic locus is a modified 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 a sPAM and/or sigRNAR in, adjacent to, or operably linked to one or both DNA junction polynucleotides of the modified 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 transgenic locus. 
     
     
         41 . The nucleic acid marker of  claim 37 , 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 a sPAM and/or sigRNAR in, adjacent to, or operably linked to one or both DNA junction polynucleotides of the modified 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 transgenic locus. 
     
     
         42 . The nucleic acid marker of  claim 37 , wherein the modified transgenic locus is a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus comprising a sPAM and/or sigRNAR in, adjacent to, or operably linked to one or both DNA junction polynucleotides of the modified 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. 
     
     
         43 . The nucleic acid marker of  claim 37 , wherein the modified transgenic locus is a GT73, HCN28, MON88302, or MS8 transgenic locus comprising a sPAM and/or sigRNAR in, adjacent to, or operably linked to one or both DNA junction polynucleotides of the modified transgenic locus. 
     
     
         44 . A processed transgenic plant product obtained from the transgenic plant part of  claim 22  or  23 , wherein the processed plant product contains a polynucleotide comprising a sPAM and/or sigRNAR in or adjacent to one or both DNA junction polynucleotides of the first, second and/or third modified transgenic locus. 
     
     
         45 . A biological sample obtained from the transgenic plant cell of  claim 20 , the transgenic plant of  claim 21 , or the transgenic plant part of  claim 22 , wherein the biological sample contains one or more polynucleotide(s) comprising the sPAM and/or sigRNAR in one or both DNA junction polynucleotides of the first, second and/or third modified transgenic locus. 
     
     
         46 . Method of detecting the edited transgenic plant genome of any one of  claims 1  to  9 , comprising the step of detecting the presence of a polynucleotide comprising one or more of said sPAMs and/or sigRNAR. 
     
     
         47 . The method of  claim 46 , wherein the polynucleotide is detected by detecting a single nucleotide polymorphism (SNP) in the sPAM and/or sigRNAR that is present in the modified transgenic locus but absent in the original transgenic locus. 
     
     
         48 . The method of  claim 46 , 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. 
     
     
         49 . A method of obtaining an edited transgenic plant genome comprising a modified transgenic locus comprising the step of introducing a first sPAM site in or adjacent to a first DNA junction polynucleotide of an original transgenic locus, wherein the sPAM site is operably linked to the first DNA junction polynucleotide. 
     
     
         50 . A method of obtaining an edited transgenic plant genome comprising a modified transgenic locus comprising the step of introducing a first and a second sPAM site in or adjacent to a first and a second DNA junction polynucleotide of an original transgenic locus, wherein the sPAM sites are operably linked to the first and the second DNA junction polynucleotide. 
     
     
         51 . The method of  claim 50 , wherein each sPAM is introduced by:
 (a) contacting the original transgenic locus with: (i) a catalytically deficient RNA dependent DNA endonuclease (cdRdDe) or RdDe nickase, wherein the cdRdDe or RdDe nickase is operably linked to a nucleobase deaminase; and (ii) a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within or adjacent to a first junction polynucleotide of the original transgenic locus; and   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the first and second sPAM.   
     
     
         52 . The method of  claim 51 , wherein the nucleobase deaminase is a cytosine deaminase or an adenine deaminase. 
     
     
         53 . The method of  claim 50 , wherein at least one sPAM is introduced by:
 (a) contacting the original transgenic locus with: (i) a Zinc Finger Nuclease or TALEN which recognizes a junction polynucleotide of the original transgenic locus or (ii) a Zinc Finger nickase or Tale nickase which recognizes a junction polynucleotide of the original transgenic locus, and optionally a donor DNA template spanning a double stranded DNA break site in the junction polynucleotide; and   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the sPAM.   
     
     
         54 . The method of  claim 50 , 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. 
     
     
         55 . The method of  claim 54 , 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. 
     
     
         56 . A method of obtaining an edited transgenic plant genome comprising a modified transgenic locus comprising the step of introducing a sigRNAR site in or adjacent to a first DNA junction polynucleotide of an original transgenic locus, wherein the sigRNAR site is operably linked to the first DNA junction polynucleotide. 
     
     
         57 . A method of obtaining an edited transgenic plant genome comprising a modified transgenic locus comprising the step of introducing a sigRNAR site in or adjacent to a first and a second DNA junction polynucleotide of an original transgenic locus, wherein the sigRNAR sites are operably linked to the first and the second DNA junction polynucleotide. 
     
     
         58 . The method of  claim 57 , wherein each sigRNAR is introduced by:
 (a) contacting the original transgenic locus with: (i) an RdRe 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 or adjacent to a first junction polynucleotide of the original transgenic locus; (ii) a guide RNA comprising an RNA equivalent of the DNA located immediately 5′ or 3′ to an original PAM site located within or adjacent to a first junction polynucleotide of the original transgenic locus; and (iii) a donor DNA template spanning a double stranded DNA break site in the junction polynucleotide comprising a heterologous crRNA (CRISPR RNA) binding sequence of the sigRNAR and optionally a PAM or sPAM site; and   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the sigRNAR site.   
     
     
         59 . The method of  claim 57 , wherein each sigRNAR is introduced by:
 (a) contacting the original transgenic locus with: (i) a Zinc Finger Nuclease or TALEN which recognizes a junction polynucleotide of the original transgenic locus or (ii) a Zinc Finger nickase or Tale nickase which recognizes a junction polynucleotide of the original transgenic locus, and a donor DNA template spanning a double stranded DNA break site in the junction polynucleotide comprising a heterologous crRNA (CRISPR RNA) binding sequence of the sigRNAR and optionally a PAM or sPAM site; and   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant comprising the sigRNAR sites.   
     
     
         60 . The method of  claim 57 , 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. 
     
     
         61 . The method of  claim 60 , 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. 
     
     
         62 . A method of excising a modified transgenic locus from an edited transgenic plant genome comprising the steps of:
 (a) contacting the edited transgenic plant genome of any one of  claims 1  to  19  with: (i) an RdDe that recognizes the first set of sPAMs, the second set of sPAMs, and/or the third set of sPAMs; and (ii) two guide RNAs (gRNAs), wherein each gRNA comprises an RNA equivalent of the DNA located immediately 5′ or 3′ to the first set of sPAMs; and,   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the modified transgenic locus flanked by the first set of sPAMs has been excised.   
     
     
         63 . A method of excising a modified transgenic locus from an edited transgenic plant genome comprising the steps of:
 (a) contacting the edited transgenic plant genome of any one of  claims 1  to  19  with: (i) an RdDe that recognizes the sPAM in a first junction polynucleotide and a pre-existing PAM or sigRNAR site in a second junction polynucleotide of a first transgenic locus; and (ii) two guide RNAs (gRNAs), wherein each gRNA comprises an RNA equivalent of the DNA located immediately 5′ or 3′ to the sPAM and pre-existing PAM or sigRNAR site; and,   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the modified transgenic locus flanked by the sPAM and the pre-existing PAM or sigRNAR site has been excised.   
     
     
         64 . The method of  claim 63 , wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising or encoding the RdDe(s) and gRNAs into a transgenic plant cell comprising the edited transgenic plant genome. 
     
     
         65 . The method of  claim 63 , wherein the transgenic plant cell is in tissue culture, in a callus culture, a plant part, or in a whole plant. 
     
     
         66 . The method of  claim 63 , wherein the transgenic plant cell is a haploid plant cell. 
     
     
         67 . A method of excising a modified transgenic locus from an edited transgenic plant genome comprising the steps of:
 (a) contacting the edited transgenic plant genome of any one of  claims 1  to  19  with: (i) an RdDe that recognizes the first set of sigRNAR sites, the second set of sigRNAR sites, and/or the third set of sigRNAR sites; and (ii) a guide RNA (gRNA) directed to the first set of sigRNAR sites; and,   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the modified transgenic locus flanked by the first set of sigRNAR sites has been excised.   
     
     
         68 . A method of excising a modified transgenic locus from an edited transgenic plant genome comprising the steps of:
 (a) contacting the edited transgenic plant genome of any one of  claims 1  to  19  with: (i) an RdDe that recognizes a sigRNAR site in a first junction polynucleotide and a pre-existing PAM or sPAM site in a second junction polynucleotide of the first transgenic locus; and (ii) a guide RNA (gRNA) directed to the first sigRNAR sites and the pre-existing PAM or sPAM site; and,   (b) selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the modified transgenic locus flanked by the sigRNAR, and pre-existing PAM or sPAM sites has been excised.   
     
     
         69 . The method of  claim 68 , wherein the edited transgenic plant genome is contacted in step (a) by introducing one or more compositions comprising or encoding the RdDe(s) and gRNAs into a transgenic plant cell comprising the edited transgenic plant genome. 
     
     
         70 . The method of  claim 68 , wherein the transgenic plant cell is in tissue culture, in a callus culture, a plant part, or in a whole plant. 
     
     
         71 . The method of  claim 68 , wherein the transgenic plant cell is a haploid plant cell. 
     
     
         72 . A method of obtaining a plant breeding line comprising:
 (a) crossing a transgenic plants comprising the edited transgenic genomes of any of  claims 1  to  19 , 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.   
     
     
         73 . The method of  claim 72 , wherein the second plant of (a) further comprises the third modified transgenic locus and wherein a progeny plant comprising the first, second, and third modified transgenic locus from the cross is selected in (b). 
     
     
         74 . The method of  claim 72  or  73 , 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. 
     
     
         75 . 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 signature protospacer adjacent motif (sPAM) sites or signature guide RNA Recognition (sigRNAR) sites are operably linked to both DNA junction polynucleotides of at least the first transgenic locus and optionally to the second transgenic loci;   (b) contacting the transgenic plant genome of the donor inbred parent plant line with: (i) at least a first guide RNA directed to genomic DNA adjacent to two sPAM sites or directed to the sigRNAR sites, wherein the sPAM or sigRNAR sites are operably linked to the first transgenic locus; and (ii) one or more RNA dependent DNA endonucleases (RdDe) which recognize the sPAM or sigRNAR sites; 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.   
     
     
         76 . The method of  claim 75 , 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. 
     
     
         77 . The method of  claim 75 , 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. 
     
     
         78 . The method of  claim 75 , 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. 
     
     
         79 . The method of  claim 75 , wherein a third transgenic locus is introgressed or introduced into the inbred germplasm to obtain a donor inbred parent plant line comprising the first, second, and third transgenic loci. 
     
     
         80 . The method of  claim 75 , further comprising contacting the transgenic plant genome with a second guide RNA directed to genomic DNA adjacent to two sPAM sites, wherein the sPAM sites are operably linked to a 5′ and a 3′ DNA junction polynucleotide of the second or third transgenic locus; and (ii) one or more RNA dependent DNA endonucleases (RdDe) which recognize the sPAM sites in step (b); and selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the second or third transgenic locus has been excised in step (c). 
     
     
         81 . The method of  claim 75 , further comprising contacting the transgenic plant genome with a second guide RNA directed to sigRNA sites which are operably linked to a 5′ and a 3′ DNA junction polynucleotide of the second or third transgenic locus; and (ii) one or more RNA dependent DNA endonucleases (RdDe) which recognize the sigRNAR sites in step (b); and selecting a transgenic plant cell, transgenic plant part, or transgenic plant wherein the second or third transgenic locus has been excised in step (c). 
     
     
         82 . The method of  claim 75 , 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. 
     
     
         83 . The method of  claim 75 , wherein the transgenic plant genome of step (b) further comprises a third transgenic plant locus wherein signature protospacer adjacent motif (sPAM) sites are operably linked to both DNA junction polynucleotides of the third transgenic locus. 
     
     
         84 . The method of  claim 75 , 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). 
     
     
         85 . The method of  claim 75 , 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). 
     
     
         86 . The method of  claim 75 , 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.   
     
     
         87 . The method of any one of  claims 75  to  86 , wherein the transgenic plant germplasm is transgenic corn plant germplasm and wherein the first, second, and/or third transgenic locus comprises a 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, and/or TC1507 transgenic locus in a transgenic corn plant genome, said modification comprising signature protospacer adjacent motif (sPAM) sites and/or sigRNAR sites which are operably linked to both DNA junction polynucleotides of the 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 transgenic locus. 
     
     
         88 . The method of any one of  claims 75  to  86 , wherein the transgenic plant germplasm is transgenic soybean plant germplasm and wherein the first, second, and/or third 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, and/or SYHT0H2 transgenic locus in a transgenic soybean plant genome, said modification comprising signature protospacer adjacent motif (sPAM) sites and/or sigRNAR sites which are operably linked to both DNA junction polynucleotides of the 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 transgenic locus. 
     
     
         89 . The method of any one of  claims 75  to  86 , wherein the transgenic plant germplasm is transgenic cotton plant germplasm and wherein the first, second, and/or third transgenic locus comprises a modification of a DAS-21023-5, DAS-24236-5, COT102, LLcotton25, MON15985, MON88701, and/or MON88913 transgenic locus in a transgenic cotton plant genome, said modification comprising signature protospacer adjacent motif (sPAM) sites and/or sigRNAR sites which are operably linked to both DNA junction polynucleotides of the 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 transgenic locus. 
     
     
         90 . The method of any one of  claims 75  to  86 , wherein the transgenic plant germplasm is transgenic canola plant germplasm and wherein the first, second, and/or third transgenic locus comprises a modification of a GT73, HCN28, MON88302, or MS8 transgenic locus in a transgenic canola plant genome, said modification comprising signature protospacer adjacent motif (sPAM) sites and/or sigRNAR sites which are operably linked to both DNA junction polynucleotides of the 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 transgenic locus.