CRISPR-Cas LOX Gene Editing for Maize Disease Resistance
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Solution Overview
Problem
Current methods for modifying genes in plants to resist ear rot and stalk rot pathogens, such as those caused by Stenocarpella maydis and Stenocarpella macrospora, often result in off-types and growth issues, necessitating the development of novel strategies for increasing resistance without adverse effects on plant development.
Innovation Solution
The use of CRISPR-Cas effector proteins and guide nucleic acids to introduce non-natural mutations in Lipoxygenase (LOX) genes in maize plants, specifically targeting sites with high sequence identity, to create null alleles or knockout mutations, thereby enhancing resistance to ear and stalk rots without introducing transgenes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Mu transposase-based gene knockouts or transgenic overexpression/knockdown are used to modify defense genes, then resistance to pathogens is improved, but plant growth and development are disrupted (off-types are produced)
Solution Approach 1:
The patent extracts and removes the harmful transgenic elements (promoters, selection markers, transposase sequences) while retaining the desired gene knockout effect. By using CRISPR-Cas to create precise mutations and then eliminating the editing machinery through segregation, the invention keeps only the beneficial resistance trait without the adverse growth effects caused by transgenic components.
Solution Approach 2:
The invention changes the method of gene modification from transgenic overexpression/knockdown to precise CRISPR-mediated mutation. This parameter change in the editing approach allows for cleaner genetic modifications that do not interfere with plant development, while still achieving the desired pathogen resistance through LOX gene disruption.
2Reliability
If transgenic methods are used to modify LOX genes, then resistance to ear rot and stalk rot is improved, but transgenes remain in the plant genome
Solution Approach 1:
The patent removes transgenic elements from the final product by using CRISPR-Cas editing followed by segregation of the editing machinery. The guide RNA and Cas protein are transiently expressed and then eliminated, leaving only the desired LOX gene mutation in the plant genome, achieving transgene-free edited plants with improved resistance.
Solution Approach 2:
The invention performs preliminary CRISPR editing to create the desired LOX mutations, then allows the transgenic components to be naturally segregated and eliminated in subsequent generations. This preliminary action followed by cleanup achieves the dual goal of improved resistance and transgene-free status.
3Reliability
If conventional gene modification approaches are used, then disease resistance is achieved, but off-types related to plant growth and development are produced
Solution Approach 1:
The patent changes the precision and specificity of gene modification by using CRISPR-Cas to create targeted mutations in LOX genes rather than using粗放 transgenic methods. This precise editing approach maintains consistent plant phenotypes while achieving disease resistance, eliminating the off-type variations caused by non-specific transgenic modifications.
Data Source
AI summary
This invention relates to compositions and methods for modifying LOX genes and inmaize plants to increase resistance to ear rot and/or stalk rot disease. The invention further relates to plants having increased resistance to ear rot and/or stalk rot produced using the methods and compositions of the invention.