Expedited breeding of transgenic crop plants by genome editing

Abstract

Methods of selectively excising transgenic loci from transgenic plants and use of such methods to facilitate plant breeding are disclosed.

1 . A method of producing an elite maize plant comprising a targeted genetic change and at least one approved transgenic locus comprising steps of:
 (i) inducing at least one targeted genetic change in the genome of the maize plant with one or more genome editing molecules in an elite maize plant comprising a first approved transgenic loci or modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA and a second approved transgenic loci or modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA;   (ii) excising a DNA segment comprising all or most of the first approved transgenic locus with genome editing molecules by (a) contacting genomic DNA of said plant with: (i) at least a first and at least a second guide RNA directed to genomic DNA adjacent to two PAM sites, wherein the PAM sites are operably linked to a 5′ and a 3′ DNA junction polynucleotide of the first approved transgenic locus; and (ii) one or more RNA dependent DNA endonucleases (RdDe) which recognize the PAM sites; and   (iii) selecting an elite maize plant wherein the first approved transgenic locus or modification thereof is excised, the second approved transgenic locus or modification thereof is present, and the targeted genetic change is present.   
     
     
         2 . The method of  claim 1 , wherein steps (i) and (ii) are performed sequentially. 
     
     
         3 . The method of  claim 1 , wherein steps (i) and (ii) are performed simultaneously. 
     
     
         4 . The method of  claim 1 , wherein the targeted genetic change confers a desirable agronomic or quality trait. 
     
     
         5 . The method of  claim 1 , wherein all of the first approved transgenic locus and one or more nucleotides of endogenous chromosomal DNA of the plant genome in the 5′ and a 3′ DNA junction polynucleotide is excised. 
     
     
         6 . The method of  claim 1  wherein all but at least one nucleotide of the heterologous DNA of the 5′ and/or 3′ DNA junction polynucleotide of the first approved transgenic locus is excised 
     
     
         7 . The method of  claim 1 , further comprising the step of obtaining the elite crop plant comprising a first approved transgenic loci and a second approved transgenic loci of step (i). 
     
     
         8 . The method of  claim 1 , wherein the first approved transgenic locus is selected from the group consisting of 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, TC1507 transgenic locus and a modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA, in a transgenic maize plant genome. 
     
     
         9 . The method of  claim 1 , wherein the second approved transgenic locus is selected from the group consisting of 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, TC1507, and a modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA, in a transgenic maize plant genome. 
     
     
         10 - 15 . (canceled) 
     
     
         16 . A method for obtaining an elite maize plant, comprising the steps of: (a) obtaining a maize plant comprising at least an approved first transgenic locus and a second approved transgenic locus; (b) introgressing the first approved transgenic locus and a second approved transgenic locus into the germplasm of the elite maize plant; (c) excising a DNA segment comprising the second approved transgenic locus from the elite maize plant of step (b) with genome editing molecules and optionally inducing at least one targeted genetic change in the genome of the maize plant of step (b) with one or more genome editing molecules; and (d) selecting an elite maize plant comprising: (i) the approved first transgenic locus and a transgenic locus excision site wherein all of the second approved transgenic locus and one or more nucleotides of endogenous chromosomal DNA of the plant genome in the 5′ and a 3′ DNA junction polynucleotide of the second approved transgenic locus is excised; and optionally (ii) the targeted genetic change. 
     
     
         17 . The method of  claim 16 , wherein the introgression comprises: (i) crossing the maize plant of (a) to a plant comprising the elite maize germplasm but lacking both the first and the second transgenic locus; (ii) selecting a progeny plant comprising the first and the second transgenic locus; (iii) backcrossing the progeny plant to a recurrent parent maize plant comprising the elite maize germplasm but lacking the first and second transgenic locus; and (iv) selecting a progeny plant comprising the first and the second transgenic locus. 
     
     
         18 - 44 . (canceled) 
     
     
         45 . The method of  claim 16 , wherein the first approved transgenic locus comprises 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, TC1507 transgenic locus, or a modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA, in a transgenic maize plant genome. 
     
     
         46 . The method of  claim 16 , wherein the second approved transgenic locus comprising a DAS-59122-7, DP-4114, MON87411, 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, TC1507 transgenic locus, or a modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA, is excised. 
     
     
         47 . The method of  claim 46 , wherein the plant further comprises a third transgenic locus comprising a selectable marker gene which confers a selectable marker trait of a DAS-59122-7, DP-4114, MON87411, 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. 
     
     
         48 . (canceled) 
     
     
         49 . A method for obtaining elite maize plant, comprising the steps of: (a) obtaining a maize plant comprising at least an approved first transgenic locus and a second approved transgenic locus; (b) introgressing the first approved transgenic locus and the second approved transgenic locus into the germplasm of the elite maize plant; (c) excising a DNA segment comprising the second approved transgenic locus from the elite maize plant of step (b) with genome editing molecules and optionally inducing at least one targeted genetic change in the genome of the maize plant of step (b) with one or more genome editing molecules; and (d) selecting an elite maize plant comprising: (i) the approved first transgenic locus and a transgenic locus excision site wherein all of the second approved transgenic locus and one or more nucleotides of endogenous chromosomal DNA of the plant genome in the 5′ and a 3′ DNA junction polynucleotide of the second approved transgenic locus is excised; and optionally (ii) the targeted genetic change. 
     
     
         50 . The method of  claim 49 , wherein the introgression comprises: (i) crossing the maize plant of (a) to a plant comprising the elite maize germplasm but lacking both the first and the second transgenic locus; (ii) selecting a progeny plant comprising the first and the second transgenic locus; (iii) backcrossing the progeny plant to a recurrent parent maize plant comprising the elite maize germplasm but lacking the first and second transgenic locus; and (iv) selecting a progeny plant comprising the first and the second transgenic locus. 
     
     
         51 - 77 . (canceled) 
     
     
         78 . The method of  claim 49 , wherein the first approved transgenic locus comprises 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, TC1507 transgenic locus, or a modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA, in a transgenic maize plant genome. 
     
     
         79 . The method of  claim 49 , wherein the second approved transgenic locus comprising a DAS-59122-7, DP-4114, MON87411, 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, TC1507 transgenic locus, or a modification thereof comprising a deletion of at least one selectable marker gene and/or non-essential DNA, is excised. 
     
     
         80 . The method of  claim 50 , wherein the plant further comprises a third transgenic locus comprising a selectable marker gene which confers a selectable marker trait of a DAS-59122-7, DP-4114, MON87411, 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.