Inir17 transgenic maize

Abstract

Transgenic INIR17 maize plants comprising an ecry3.1Ab expression cassette linked to a secondary nopaline synthase terminator element which lack a selectable marker gene and/or which comprise modifications that provide for facile excision of the INIR17 transgenic locus from the maize plant genome are provided. Genomic DNA of INIR17 transgenic plants, detection of INIR17 plants and products thereof, methods of making INIR17 plants, and use of INIR17 plants to facilitate breeding are disclosed.

What is claimed is: 
     
         1 . A transgenic maize plant cell comprising a transgenic locus comprising the DNA molecule of SEQ ID NO: 33, 29, 30, 32, 26, 27, 34, 36, or an allelic variant thereof. 
     
     
         2 . The transgenic maize cell of  claim 1 , wherein the transgenic locus comprises the DNA molecule of SEQ ID NO: 33, 29, 30, or an allelic variant thereof. 
     
     
         3 . The transgenic maize cell of  claim 1 , wherein the transgenic locus comprises the DNA molecule of SEQ ID NO: 33. 
     
     
         4 . A transgenic maize plant seed comprising a transgenic locus comprising the DNA molecule of SEQ ID NO: 33, 29, 30, 32, 26, 27, 34, 36, or an allelic variant thereof. 
     
     
         5 . A transgenic maize plant comprising a transgenic locus comprising the DNA molecule of SEQ ID NO: 33, 29, 30, 32, 26, 27, 34, 36, or an allelic variant thereof. 
     
     
         6 . A method for obtaining a bulked population of seed comprising selfing the transgenic maize plant of  claim 5  and harvesting transgenic seed comprising the transgenic locus comprising the DNA molecule of SEQ ID NO: 33, 29, 30, 32, 26, 27, 34, 36, or an allelic variant thereof. 
     
     
         7 . A method for obtaining a bulked population of inbred seed comprising selfing the transgenic maize plant of  claim 5  and harvesting seed comprising the INIR17 transgenic locus from the selfed maize plant. 
     
     
         8 . A method of obtaining hybrid maize seed comprising crossing the transgenic maize plant of  claim 5  to a second maize plant which is genetically distinct from the first maize plant and harvesting seed comprising the INIR17 transgenic locus from the cross. 
     
     
         9 . A DNA molecule comprising SEQ ID NO: 16, 26, 27, 28, 29, 32, 33, 34, 36, 38, 39, or 40. 
     
     
         10 . A processed transgenic maize plant product comprising the DNA molecule of  claim 9 . 
     
     
         11 . A biological sample containing the DNA molecule of  claim 9 . 
     
     
         12 . A nucleic acid molecule adapted for detection of genomic DNA comprising the DNA molecule of  claim 9 , wherein said nucleic acid molecule optionally comprises a detectable label. 
     
     
         13 . A method of detecting a maize plant cell comprising the transgenic locus of  claim 1 , comprising the step of detecting a DNA molecule comprising SEQ ID NO: 16, 26, 27, 28, 29, 32, 33, 34, 36, 38, 39, or 40. 
     
     
         14 . A method of excising a transgenic locus comprising a DNA molecule of SEQ ID NO: 33, 29, 30, or an allelic variant thereof from the genome of a maize plant cell, comprising the steps of:
 (a) contacting the genome of the maize plant cell 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 originator guide RNA recognition site (OgRRS) and the cognate guide RNA recognition site (CgRRS) of the transgenic locus; 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 transgenic locus flanked by the OgRRS and the CgRRS has been excised.   
     
     
         15 . The method of  claim 14 , wherein the OgRRS is located in a 3′ flanking DNA junction polynucleotide and comprises SEQ ID NO: 20 and wherein the CgRRS comprises an insertion or substitution of SEQ ID NO:17 in a 5′ junction polynucleotide of said transgenic locus. 
     
     
         16 . The method of  claim 14 , wherein the insertion and/or substitution is in a 5′ junction polynucleotide of the transgenic locus corresponding to at least one of: (i) nucleotides 1350 to 1356 of SEQ ID NO:1; or (ii) nucleotides 1336 to 1342 of SEQ ID NO: 1. 
     
     
         17 . The method of  claim 14 , wherein the CgRRS comprises the DNA molecule set forth in SEQ ID NO:16. 
     
     
         18 . A method of modifying a transgenic maize plant cell comprising: obtaining a 5307 maize event plant cell, a representative sample of which was deposited at the ATCC under accession No. PTA-9561, comprising a nucleotide sequence comprising a CMP promoter, a eCry3.1Ab coding region which is operably linked to said promoter, a first nopaline synthase (NOS) terminator element which is operably linked to said eCry3.1Ab coding region, a ZmUbiInt promoter and an operably linked phosphomannose isomerase coding region, and a second NOS terminator element; and modifying said nucleotide sequence to eliminate functionality of said phosphomannose isomerase coding region and/or to substantially, essentially, or completely remove said phosphomannose isomerase coding region, and optionally to eliminate functionality of, or substantially, essentially, or completely remove, said first NOS terminator, said ZmUbiInt promoter, and said operably linked phosphomannose isomerase coding region. 
     
     
         19 . A method of modifying a transgenic maize plant cell comprising: obtaining a 5307 maize event plant cell, a representative sample of which was deposited at the ATCC under accession No. PTA-9561, comprising a nucleotide sequence comprising a 5′ junction polynucleotide, a CMP promoter, a eCry3.1Ab coding region which is operably linked to said promoter, a first nopaline synthase (NOS) terminator element which is operably linked to said eCry3.1Ab coding region, a ZmUbiInt promoter and an operably linked phosphomannose isomerase coding region, and a second NOS terminator element; and modifying said nucleotide sequence to: (i) substantially, essentially, or completely remove said first NOS terminator, said ZmUbiInt promoter, and said operably linked phosphomannose isomerase coding region; and (ii) delete and/or substitute one or more nucleotides of said 5′ junction polynucleotide, optionally wherein one or more nucleotides or a polynucleotide sequence comprising a CgRRS are inserted into said junction polynucleotide. 
     
     
         20 . A method of making transgenic maize plant cell comprising an INIR17 transgenic locus comprising:
 (a) contacting the transgenic plant genome of a maize 5307 plant cell with: (i) a first set of gene editing molecules comprising a first site-specific nuclease which introduces at least one first double stranded DNA break in a 5′ junction polynucleotide of a 5307 transgenic locus; and (ii) a second set of gene editing molecules comprising a second site-specific nuclease which introduces a second double stranded DNA break between the eCry3.1Ab coding sequence and the first nopaline synthase (NOS) terminator of said 5307 transgenic locus and a third site specific nuclease which introduces a third double stranded DNA break between the phosphomannose isomerase coding region and DNA encoding the second nopaline synthase (nos) terminator element of said 5307 transgenic locus; and   (b) selecting a transgenic maize plant cell, transgenic maize callus, and/or a transgenic maize plant comprising an INIR17 transgenic locus wherein one or more nucleotides of said 5′ junction polynucleotide have been deleted and/or substituted, wherein the CMP promoter, the eCry3.1Ab coding region which is operably linked to the CMP promoter, and the second NOS terminator element of said 5307 transgenic locus are present, and wherein DNA of said 5307 transgenic locus comprising the first NOS terminator, the ZmUbiInt promoter and the phosphomannose isomerase coding region is absent, thereby making a transgenic maize plant cell comprising an INIR17 transgenic locus.   
     
     
         21 . The method of  claim 20 , comprising:
 (a) contacting the transgenic plant genome of a maize 5307 plant cell with: (i) a first set of gene editing molecules comprising a first site-specific nuclease which introduces at least one first double stranded DNA break between nucleotide residues corresponding to nucleotides 1350 to 1356 of SEQ ID NO:1; and/or nucleotides 1336 to 1342 of SEQ ID NO: 1; and (ii) a second set of gene editing molecules comprising a second site-specific nuclease which introduces a second double stranded DNA break between nucleotide residues corresponding to nucleotide number 3866 to 3895 of SEQ ID NO:1 and a third site specific nuclease which introduces a third double stranded DNA break between nucleotide residues corresponding to nucleotide number 7356 to 7415 of SEQ ID NO:1; and   (b) selecting a transgenic maize plant cell, transgenic maize plant callus, and/or a transgenic maize plant wherein one or more nucleotides corresponding to nucleotide number 1336 to 1356 of SEQ ID NO:1 have been deleted and/or substituted, wherein nucleotides corresponding to at least nucleotide number 4164 to 7355 of SEQ ID NO:1 have been deleted and/or replaced, and wherein nucleotides corresponding to at least nucleotide number 1360 to 3865 and 7415 to 8865 of SEQ ID NO:1 are retained.   
     
     
         22 . The method of  claim 20 , further comprising contacting the transgenic plant genome of the maize 5307 plant cell with a donor DNA template comprising a cognate guide RNA recognition site (CgRRS), wherein said CgRRS optionally comprises a polynucleotide set forth in SEQ ID NO:17; and selecting a transgenic plant cell wherein said CgRRS has integrated into and/or replaced one or more nucleotides corresponding to at least one of nucleotides of SEQ ID NO:1. 
     
     
         23 . The method of  claim 20 , wherein the gene editing molecules comprise: (i) a zinc finger nuclease; (ii) a TALEN; and/or (iii) an RNA dependent DNA endonuclease (RdDe) and a guide RNA. 
     
     
         24 . The method of  claim 23 , wherein the RNA dependent DNA endonuclease (RdDe) comprises a Cas12a RdDe and wherein the guide RNA of said first set of gene editing molecules comprises SEQ ID NO:8, 9, 10, and/or 11, the guide RNA of said second set of gene-editing molecules comprises SEQ ID NO:12, and the guide RNA of said third set of gene-editing molecules comprises SEQ ID NO:13. 
     
     
         25 . The method of any one of  claim 18 ,  19 ,  20 , or  21 , further comprising the step of regenerating transgenic maize plant callus and/or a transgenic maize plant comprising the modification or the INIR17 transgenic locus from said transgenic maize plant cell selected in step (c). 
     
     
         26 . The method of any one of  claim 18 ,  19 ,  20 , or  21 , further comprising the step of harvesting a transgenic maize plant seed comprising the modification or the INIR17 transgenic locus from the transgenic maize plant comprising the modification or the INIR17 transgenic locus.