Haploid Inducer Mediates CRISPR Delivery to Recalcitrant Crops
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Solution Overview
Problem
Current methods for introducing gene-editing machinery into plants are limited by tissue culture recalcitrance, particularly in transformation-recalcitrant crops or varieties, which are valuable but cannot be transformed directly.
Innovation Solution
The method involves using haploid inducing lines to transiently introduce gene-editing machinery into plants during haploid induction, allowing for simultaneous editing and haploid induction in a wide range of crops without the need for tissue culture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If transformation methods (Agrobacterium or biolistic transformation) are used to introduce gene-editing machinery into plants, then gene editing capability is achieved, but the method fails for transformation-recalcitrant crops or varieties due to tissue culture dependency
Solution Approach 1:
The patent uses a haploid inducer parent as an intermediary carrier to deliver gene-editing machinery (CRISPR-Cas9 system) to the target plant. Instead of directly transforming the recalcitrant crop, the editing machinery is introduced into the haploid inducer parent, which then naturally delivers it during the haploid induction process through pollen transmission and fertilization, bypassing the need for direct transformation of the target crop
Solution Approach 2:
The gene-editing machinery is preliminarily introduced into the haploid inducer parent before the actual gene editing of the target crop. This preliminary action allows the editing machinery to be prepared and optimized in a transformable line, and then naturally transmitted during the haploid induction process, eliminating the need for direct transformation of the recalcitrant crop
2Adaptability or versatility
If trait introgression is used to introduce edits into recalcitrant varieties, then gene editing is achieved, but the process is expensive, laborious, time-consuming, and results in genetic linkage impurity
Solution Approach 1:
The patent extracts only the essential gene-editing machinery (CRISPR-Cas9 system) from the complex trait introgression process. By delivering just the editing components through the haploid inducer parent, the method eliminates the need for lengthy backcrossing and selection processes required in traditional trait introgression, achieving rapid editing without genetic linkage impurity
Solution Approach 2:
The patent uses the haploid induction process to create a copy of the target plant's genome that has been edited by the CRISPR-Cas9 system delivered through the haploid inducer parent. This copying mechanism produces edited plants directly without requiring multiple generations of breeding, dramatically reducing the time and labor compared to traditional trait introgression
3Ease of operation
If floral dipping transformation is used for Arabidopsis, then gene editing machinery can be introduced transiently, but it is difficult to ensure edits end up in germ-line cells for crop plants
Solution Approach 1:
The patent utilizes the natural self-service mechanism of haploid induction, where the haploid inducer parent's pollen naturally delivers the gene-editing machinery to the target plant during fertilization. This natural delivery mechanism ensures that the editing machinery reaches the germ-line cells directly, as the pollen tube naturally targets the embryo sac and delivers contents to the egg cell and surrounding cells, including germ-line precursors
Solution Approach 2:
The patent creates a universal system where the haploid inducer parent can deliver gene-editing machinery to multiple different target crops and varieties. The same haploid inducer parent with integrated CRISPR-Cas9 system can be used across different species and varieties, providing a universal solution that works for both transformable and transformation-recalcitrant plants, ensuring reliable germ-line editing across diverse crops
Data Source
AI summary
The presently disclosed subject matter relates to using a haploid inducing line (whether existing or created) and transforming the haploid line so that it encodes cellular machinery capable of editing genes. The transformed haploid inducing line is used as a parent in a cross between two plants. During pollination, the parental gametes fuse to form an embryo; and the gene editing machinery is also delivered to the embryo at this time. During embryonic development, one set of parental chromosomes are lost, and the gene editing machinery operates on the remaining set of chromosomes. Thus, at least one haploid progeny with edited genes is produced from the cross.