CRISPR-Cas9 Wx1 Gene Knockout for High Amylopectin Maize
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Solution Overview
Problem
Conventional methods for producing Waxy maize are cumbersome and resource-intensive, often resulting in yield penalties and quality issues due to the recessive nature of Waxy mutant alleles and incomplete penetrance, making it difficult to efficiently breed Waxy maize with high amylopectin content.
Innovation Solution
The use of CRISPR-associated nucleases, such as Cas9, to introduce double-strand breaks in the Wx1 gene locus of maize plant cells, allowing for the generation of maize plants with increased amylopectin content by knocking out the Wx1 gene function, thereby producing Waxy maize with improved efficiency and yield stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional breeding methods are used to introgress Waxy mutant alleles into elite maize lines, then Waxy maize with high amylopectin content can be produced, but the process is cumbersome and resource-intensive, resulting in yield penalties and quality issues
Solution Approach 1:
The patent replaces conventional mechanical breeding methods with CRISPR/Cas9 genome editing technology. Instead of traditional cross-breeding and selection processes, the invention uses targeted molecular editing to knock out the Wx1 gene, achieving precise and efficient modification of maize genomes to produce Waxy maize with high amylopectin content without the limitations of recessive allele inheritance
Solution Approach 2:
The invention changes the fundamental approach from breeding based on allele inheritance patterns to direct genomic parameter modification. By using CRISPR/Cas9 to create specific knockout mutations in the Wx1 gene, the patent directly alters the genetic parameter controlling starch composition, enabling dominant expression of the Waxy trait and eliminating yield penalties associated with conventional breeding
2Quantity of substance
If Waxy mutant alleles are introgressed into elite maize lines via conventional breeding, then Waxy maize can be produced, but yield penalty is associated with the introgression process
Solution Approach 1:
The patent replaces conventional breeding mechanics with precise genome editing. By using CRISPR/Cas9 to directly knockout the Wx1 gene in elite maize lines, the invention eliminates the need for multi-generational breeding and selection, thereby avoiding the yield penalties and instability associated with introgressing recessive mutant alleles through traditional cross-breeding programs
3Quantity of substance
If conventional breeding methods are used to produce Waxy maize, then high amylopectin content can be achieved, but the trait exhibits incomplete penetrance and quality issues
Solution Approach 1:
The patent replaces imprecise conventional breeding with precise CRISPR/Cas9 genome editing. By targeting specific sequences in the Wx1 gene and creating defined knockout mutations, the invention achieves complete and consistent penetrance of the Waxy trait, eliminating the incomplete penetrance and quality variability inherent in traditional breeding approaches
Solution Approach 2:
The invention segments the Wx1 gene into specific targetable sequences using CRISPR guide RNA design. By dividing the gene into discrete target sites that can be precisely cleaved and disrupted, the patent ensures complete and uniform knockout across all cells, achieving full trait penetrance and consistent quality in the resulting Waxy maize
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 method enables the efficient production of Waxy maize with higher amylopectin content, reducing yield drag and quality issues associated with traditional breeding methods, while maintaining similar yields to non-Waxy hybrids.
Implementation Method 1
The use of CRISPR-associated nucleases, such as Cas9, to introduce double-strand breaks in the Wx1 gene locus of maize plant cells, allowing for the generation of maize plants with increased amylopectin content by knocking out the Wx1 gene function
Data Source
AI summary
The present disclosure involves the production of Waxy maize. Compositions and methods are provided for knocking out expression of the Waxy (Wx1) gene in maize by making double strand breaks at one or more target sites in an endogenous WX1 encoding sequence. Some methods employ one or more guide polynucleotides and a Cas endonuclease, wherein Cas endonuclease is guided by the one or more guide polynucleotides to recognize and introduce double strand breaks at specific target sites in and around the Wx1 gene. Also provided are compositions and methods for the production of Waxy maize plant cells, plant explants, seeds and grain.