INIR12 TRANSGENIC MAIZE
Transgenic INIR12 maize plants comprising a vip3Aa19 or vip3Aa20 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 INIR12 transgenic locus from the maize plant genome are provided. Genomic DNA of INIR12 transgenic plants, detection of INIR12 plants and products thereof, methods of making INIR12 plants, and use of INIR12 plants to facilitate breeding are disclosed.
1 - 43 . (canceled)
44 . A DNA molecule comprising SEQ ID NO: 48, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 37, 39, 40, 41, 42, 43, 44, 45, 47, 49, or 50-167.
45 . A processed transgenic maize plant product, transgenic maize plant cell, transgenic maize plant callus, and/or a transgenic maize plant comprising the DNA molecule of claim 44 .
46 . A biological sample comprising the DNA molecule of claim 44 .
47 . A nucleic acid molecule adapted for detection of genomic DNA comprising the DNA molecule of claim 44 , wherein the nucleic acid molecule optionally comprises a detectable label.
48 . A method of detecting a maize plant cell comprising the DNA molecule of claim 44 , comprising a step of detecting a DNA molecule comprising SEQ ID NO: 48, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 37, 39, 40, 41, 42, 43, 44, 45, 47, 49, or 50-167.
49 . A method of excising an INIR12 transgenic locus from the genome of a transgenic maize plant cell comprising the DNA molecule of claim 44 , the method comprising:
(a) contacting the edited transgenic plant genome of the plant cell of the transgenic 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 an originator guide RNA recognition site (OgRRS) and the 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 nucleotide sequence or INIR12 transgenic locus flanked by the OgRRS and the CgRRS has been excised.
50 . The method of claim 49 , wherein the OgRRS is located in a 3′ flanking DNA junction polynucleotide and comprises SEQ ID NO: 26, 27, or 28 and wherein the CgRRS comprises an insertion or substitution of SEQ ID NO: 26, 27, or 28 in a 5′ junction polynucleotide of said nucleotide sequence or said INIR12 transgenic locus.
51 . The method of claim 50 , wherein the insertion and/or substitution is in a 5′ junction polynucleotide of the INIR12 transgenic locus corresponding to at least one of nucleotides 1079 to 1098 of SEQ ID NO: 1 or SEQ ID NO:46.
52 . A method of modifying the transgenic maize plant cell of claim 45 , the method comprising:
obtaining a MIR 162 maize event plant cell, a representative sample of which was deposited at the ATCC under accession No. PTA-8166, comprising a nucleotide sequence comprising a first ZmUbiInt promoter, a vip3Aa19 or vip3Aa20 coding region which is operably linked to said promoter, a CaMV 35S terminator element which is operably linked to said vip3 Aa19 or vip3 Aa20 coding region, a second ZmUbiInt promoter and an operably linked phosphomannose isomerase coding region, and a nopaline synthase 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 second ZmUbiInt promoter.
53 . A method of modifying the transgenic maize plant cell of claim 45 , the method comprising:
obtaining a MIR 162 maize event plant cell, a representative sample of which was deposited at the ATCC under accession No. PTA-8166, comprising a nucleotide sequence comprising a first ZmUbiInt promoter, a vip3Aa19 or vip3 Aa20 coding region which is operably linked to said promoter, a CaMV 35S terminator element which is operably linked to said vip3 Aa19 or vip3 Aa20 coding region, a second ZmUbiInt promoter and an operably linked phosphomannose isomerase coding region, and a nopaline synthase terminator element; and
modifying said nucleotide sequence to substantially, essentially, or completely remove said phosphomannose isomerase coding region, and optionally substantially, essentially, or completely remove said second ZmUniInt promoter.
54 . A method of making the transgenic maize plant cell of claim 45 , the method comprising:
(a) contacting the transgenic plant genome of a maize MIR162 plant cell with: (i) a first set of gene editing molecules comprising a first site-specific nuclease which introduces a first double stranded DNA break in a 5′ junction polynucleotide of an MIR162 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 CaMV35S terminator element and the ZmUbi promoter of said MIR162 transgenic locus which is operably linked to DNA encoding a phosphomannose isomerase (pmi) and a third site specific nuclease which introduces a third double stranded DNA break between the DNA encoding the pmi and DNA encoding the nopaline synthase (nos) terminator element of said MIR162 transgenic locus; and
(b) selecting a transgenic maize plant cell, transgenic maize callus, and/or a transgenic maize plant comprising an INIR12 transgenic locus wherein one or more nucleotides of said 5′ junction polynucleotide have been deleted and/or substituted, wherein the first ZmUbiInt promoter, the vip3Aa19 or vip3Aa20 coding region which is operably linked to the first ZmUbiInt promoter, the CaMV 35S terminator element which is operably linked to said vip3 Aa19 or vip3 Aa20 coding region, and the nos terminator element of said MIR162 transgenic locus are present, and wherein DNA of said MIR162 transgenic locus comprising a second ZmUbiInt promoter and an operably linked phosphomannose isomerase coding region is absent, thereby making a transgenic maize plant cell comprising an INIR12 transgenic locus.
55 . The method of claim 54 , comprising:
(a) contacting the transgenic plant genome of a maize MIR162 plant cell with: (i) a first set of gene editing molecules comprising a first site-specific nuclease which introduces a first double stranded DNA break between nucleotide residues corresponding to nucleotide number 1079 to 1098 of SEQ ID NO:1 or SEQ ID NO:46; 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 5838 to 5858 of SEQ ID NO: 1 or SEQ ID NO:46 and a third site specific nuclease which introduces a third double stranded DNA break between nucleotide residues corresponding to nucleotide number 9040 to 9105 of SEQ ID NO: 1 or SEQ ID NO:46; 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 1081 to 1104 of SEQ ID NO:1 or SEQ ID NO:46 have been deleted and/or substituted, wherein nucleotides corresponding to at least nucleotide number 5858 to 9040 of SEQ ID NO: 1 or SEQ ID NO:46 have been deleted and/or replaced, and wherein nucleotides corresponding to at least nucleotide number 1105 to 5837 of SEQ ID NO:1 or SEQ ID NO:46 are retained.
56 . The method of claim 54 , further comprising contacting the transgenic plant genome of the maize MIR162 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: 26, 27, 28, or 37; 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 1079 to 1098 of SEQ ID NO:1 or SEQ ID NO:46.
57 . The method of claim 52 , 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; and optionally 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: 20, the guide RNA of said second set of gene-editing molecules comprises SEQ ID NO: 21, and the guide RNA of said third set of gene-editing molecules comprises SEQ ID NO: 23.
58 . The method of claim 52 , further comprising the step of regenerating transgenic maize plant callus and/or a transgenic maize plant comprising the modification or the INIR12 transgenic locus from said transgenic maize plant cell selected in step (c).
59 . The method of claim 58 , further comprising the step of harvesting a transgenic maize plant seed comprising the modification or the INIR12 transgenic locus from the transgenic maize plant comprising the modification or the INIR12 transgenic locus.
60 . A transgenic maize plant cell, plant callus, plant seed, or plant part comprising a modification or an INIR12 transgenic locus made by the method of claim 52 .
61 . A method of using the processed transgenic maize plant product, transgenic maize plant cell, transgenic maize plant callus, and/or a transgenic maize plant of claim 45 for collecting nucleic acid analysis data; wherein said method comprises: (a) isolating the nucleic acids from the maize plant cell, the maize plant callus, the maize plant, the maize plant part, or the maize plant seed of and analyzing said nucleic acids, and (c) recording data based on the analysis of the nucleic acids; wherein the nucleic acid analysis data are optionally nucleic acid sequence data or nucleic acid abundance data.
62 . A method of collecting nucleic acid analysis data comprising: (a) isolating nucleic acids from the processed transgenic maize plant product, transgenic maize plant cell, transgenic maize plant callus, and/or a transgenic maize plant of claim 45 ; (b) analyzing said nucleic acids; and (c) recording data based on the analysis of the nucleic acids; wherein the nucleic acid analysis data are optionally nucleic acid sequence data or nucleic acid abundance data.
63 . A method of plant breeding comprising: (a) isolating nucleic acids from the processed transgenic maize plant product, transgenic maize plant cell, transgenic maize plant callus, and/or a transgenic maize plant of claim 45 ; (b) identifying one or more nucleic acid polymorphisms from the isolated nucleic acids; and (c) selecting a plant having one or more of the identified nucleic acid polymorphisms.