CRISPR Inverted Repeat Alleles for Dominant Gene Down-Regulation
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Solution Overview
Problem
Current methods struggle to down-regulate gene expression in hybrid plants effectively, as dominant alleles for desirable traits are often recessive, making it challenging to achieve knockdown of gene expression in hybrid crops.
Innovation Solution
Introducing an inverted repeat sequence into a gene encoding a polypeptide of interest in a plant cell using genome modification techniques like CRISPR-Cas endonuclease, which is operably linked to either the endogenous or heterologous promoter, to reduce gene expression by creating a targeted genetic modification present on only one allele.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mutations conferring knockouts and alleles with reduced gene expression are used, then gene expression can be down-regulated, but the alleles are recessive requiring both parents to carry the favorable allele
Solution Approach 1:
The patent applies inversion by inserting an inverted repeat sequence into the gene, where the sequence is transcribed and then inverted back to its original orientation during RNA processing. This creates a dominant effect because the inverted repeat interferes with the wild-type allele's expression through mechanisms like RNA interference or transcriptional interference, allowing down-regulation with only one parent carrying the modification.
2Adaptability or versatility
If dominant alleles are used for desirable traits, then only one parent needs to carry the favorable allele, but mutations conferring knockouts are typically recessive
Solution Approach 1:
The inverted repeat sequence acts as an intermediary element that mediates between the genetic modification and the gene expression regulation. When transcribed, it produces an inverted repeat RNA that interferes with the wild-type allele through RNA interference or transcriptional interference mechanisms, enabling dominant down-regulation without requiring both parents to carry the modification.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for the significant decrease in expression of specific polypeptides in hybrid plants, enabling the regulation of traits such as agronomic enhancements, resistance, and sterility, by generating hybrid plants with the desired genetic modification in at least one allele, thereby addressing the challenge of dominant alleles.
Implementation Method 1
a region of the gene encoding the polypeptide of interest comprising a nucleotide sequence that hybridizes to the inverted repeat sequence
Implementation Method 2
CRISPR-Cas endonuclease, which is operably linked to either the endogenous or heterologous promoter
Data Source
AI summary
The disclosure provides plant, plant parts, plant cells, seed and grain containing a targeted genetic modification that inserts an inverted repeat sequence into a gene. The disclosure further provides hybrid plants that contain the inverted repeat sequence in a single allele of the gene. Further provided are methods for decreasing expression of a gene encoding a polypeptide of interest in a hybrid plant by crossing a plant comprising the targeted genetic modification with a plant not comprising the genetic modification to produce a hybrid plant that contains the inverted repeat sequence in a single allele of the gene.