Heterologous integration of regulatory elements to alter gene expression in wheat cells and wheat plants
Inventors
Cermák, Tomá{hacek over (s)} • Flavell, Richard Bailey • KOCK, Michael Andreas • Konecna, Eva • Niu, Yajie • Nuccio, Michael Lee • Raji, Jennifer • Shultz, Randall William • Sosso, Davide • Unson, Maria Margarita D. • Casey, Jr., John P. • Jones, Jonathan Dallas George
Assignees
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Publication Number
US-11866719-B1
Publication Date
2024-01-09
Expiration Date
Abstract
The disclosure relates to precise modifications in wheat plant genomes including the heterologous integration of nucleic acid sequences that include a regulatory element at a predetermined locus. Also provided are wheat cells, wheat plants, wheat seeds, and processed wheat products comprising the modified wheat genomes.
Core Innovation
The invention describes a wheat plant, or a cell thereof, having a modified genome that results in modified architecture compared to an unmodified wheat plant having a reference genome. The modified genome differs from the reference genome by at least one predetermined modification of a gene, comprising heterologous integration of a nucleic acid sequence encoded by at least one donor polynucleotide molecule that includes a regulatory element at a predetermined locus between 1 and 1000 nucleotides upstream of the start codon of the coding sequence of the gene in the reference genome.
The regulatory element comprises SEQ ID NO: 231. The predetermined modification results in increased expression of the gene relative to expression of the gene in the reference genome, and the disclosure describes precise predetermined, non-random heterologous integration of non-coding regulatory elements at defined loci.
The described genome engineering is characterized across wheat types and genomic constitutions including allotetraploid A/B and allohexaploid A/B/D compositions, including specified modification patterns of homeoalleles. The modified genome is also framed by high genome identity and minimal off-target genetic and epigenetic variation such as limited DNA methylation differences.
Claims Coverage
The independent claim covers a wheat plant, or a cell thereof, with a modified genome that produces modified architecture through heterologous integration of a donor-encoded regulatory element at a defined upstream locus, leading to increased expression of the modified gene. Dependent claims further constrain genome identity, DNA methylation differences, non-coding-only changes, polyploid genome constitution and homeoallele modification patterns, and sequence identity relationships.
Heterologous upstream regulatory-element integration for increased gene expression
The predetermined modification comprises heterologous integration of a nucleic acid sequence encoded by at least one donor polynucleotide molecule and comprising a regulatory element located 1-1000 nucleotides upstream of the gene start codon, and the regulatory element comprises SEQ ID NO: 231, resulting in increased expression of the gene relative to the reference.
Modified architecture relative to an unmodified reference-genome wheat
The modified genome results in the wheat plant exhibiting modified architecture in comparison to an unmodified wheat plant having a reference genome.
Stringent genome similarity to the reference genome
The wheat plant includes a modified genome that is more than 99.9% identical to the reference genome.
Limiting DNA methylation differences on the chromosome containing the gene of interest
The modified genome shows a DNA methylation difference relative to a reference genome of less than 0.01%, 0.005%, or 0.001% within the portion of the genome contained on that chromosome.
Allotetraploid A/B homeoallele modification patterns
The wheat plant is an allotetraploid with a reference genome containing A and B genomes, and the predetermined gene modification alters homeoalleles of a gene of interest in the A genome and/or B genome.
Non-coding-only predetermined modifications
The predetermined modification changes only non-coding sequences of the reference genome.
Sequence/protein identity constraints using SEQ ID NOs
The gene encodes a protein with at least 95% identity to SEQ ID NO: 816, or is the genomic sequence of SEQ ID NO: 1100.
Across the claim set, the core scope is a wheat plant, or a cell thereof, with a modified genome that changes wheat architecture versus a reference by heterologous integration of a donor-encoded nucleic acid containing SEQ ID NO: 231 as a regulatory element upstream of a gene start codon to increase gene expression. Dependent refinements further narrow the invention by imposing quantitative identity and methylation constraints, restricting modifications to non-coding sequence changes, specifying allotetraploid A/B homeoallele modification patterns, and requiring specific gene/protein sequence identity relationships.
Stated Advantages
Modified architecture compared to an unmodified wheat plant having a reference genome.
Increased expression of the gene relative to expression of the gene in the reference genome.
Minimized off-target genetic and epigenetic variation, including limited DNA methylation differences relative to a reference genome.
Stacking of modifications across multiple wheat homeoalleles/genomes.
Altered trait phenotypes including flowering time, photosynthesis, abiotic stress tolerance, disease resistance, and senescence.
Documented Applications
Wheat plants, cells, seeds, progeny, processed wheat products, and harvested wheat products.
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