Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof
Inventors
Cigan, Andrew Mark • Falco, Saverio Carl • Lassner, Michael • Liu, Zhan-Bin • Svitashev, Sergei
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12428645-B2
Publication Date
2025-09-30
Expiration Date
Abstract
Compositions and methods are provided for genome modification of a target sequence in the genome of a plant or plant cell, without incorporating a selectable transgene marker. The methods and compositions employ a guide polynucleotide/Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed, without incorporating a selectable transgene marker. Once a genomic target site is identified, a variety of methods can be employed to further modify the target sites such that they contain a variety of polynucleotides of interest. Breeding methods and methods for selecting plants utilizing a guide polynucleotide/Cas endonuclease system are also disclosed. Compositions and methods are also provided for editing a nucleotide sequence in the genome of a cell, without incorporating a selectable transgene marker.
Core Innovation
The invention relates to introducing a polynucleotide of interest into a plant genome without introducing an exogenous selectable marker. A plant cell is provided with a Cas endonuclease and guide polynucleotides capable of forming guide/Cas complexes that introduce double strand breaks at a first target site in or near an endogenous EPSPS gene and at a second target site at a different genomic locus.
The disclosed genome engineering includes a polynucleotide modification template that causes deletion of the native promoter of the endogenous EPSPS gene with insertion of a heterologous promoter and at least one nucleotide modification of the EPSPS gene. These modifications render the endogenous EPSPS gene capable of conferring herbicide resistance to the plant cell.
A second guide/Cas complex introduces a double strand break at the second target site, and the second polynucleotide modification template comprises at least one polynucleotide of interest to be introduced into the plant genome. The approach supports targeted modification at multiple loci while maintaining a selection basis through the engineered EPSPS-related herbicide resistance.
Claims Coverage
The consolidated content provides one independent claim with four inventive features: marker-free plant genome modification, two guide RNA/Cas endonuclease complexes at two different genomic loci, EPSPS promoter deletion with heterologous promoter insertion and nucleotide modification for herbicide resistance, and introduction of a polynucleotide of interest with a second template.
Marker-free polynucleotide introduction into a plant genome
A method for introducing a polynucleotide of interest into a plant genome without introducing an exogenous selectable marker.
Two guide RNA/Cas endonuclease complexes for two different genomic loci
Providing a first guide RNA, a first polynucleotide modification template, a second guide RNA, a second polynucleotide modification template, and a Cas endonuclease to a plant cell, wherein the first guide RNA and Cas endonuclease introduce a double strand break at a first target site and the second guide RNA and Cas endonuclease introduce a double strand break at a second target site, with the first target site and the second target site located at two different genomic loci.
EPSPS native promoter deletion with heterologous promoter insertion and nucleotide modification for herbicide resistance
The first polynucleotide modification template causes a deletion of the native promoter of the endogenous EPSPS gene with the insertion of a heterologous promoter and at least one nucleotide modification of said endogenous gene to render said endogenous gene capable of conferring herbicide resistance to a plant cell.
Second template carrying the polynucleotide of interest at a different locus
The second polynucleotide modification template comprises at least one polynucleotide of interest to be introduced into said plant genome at the second target site.
The core coverage is directed to marker-free plant genome modification using two guide RNA/Cas endonuclease complexes at two different genomic loci, with one locus engineered through EPSPS promoter deletion plus heterologous promoter insertion and nucleotide modifications for herbicide resistance, and the other locus receiving at least one polynucleotide of interest.
Stated Advantages
Introducing a polynucleotide of interest into a plant genome without introducing an exogenous selectable marker.
Rendering the endogenous EPSPS gene capable of conferring herbicide resistance to a plant cell.
Maintaining a selection basis through the engineered EPSPS-related herbicide resistance.
Documented Applications
Herbicide resistance modification involving an EPSPS gene.
Introduction of a polynucleotide of interest into a plant genome at a separate genomic locus using the second polynucleotide modification template.
Selection of herbicide-resistant plant cells carrying the introduced polynucleotide of interest.
Multiplexed guide RNA/Cas9-mediated targeted insertion and mutagenesis in plant genomes, including examples in maize and stable transformation concepts in soybean.
Interested in licensing this patent?