Haploid Induction with Pollen-Mediated Gene Editing
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Solution Overview
Problem
Existing methods for gene editing in recalcitrant crop varieties are laborious, time-consuming, and introduce genetic impurities due to linkage, while transient methods in growing plants face challenges in ensuring edits are passed to progeny.
Innovation Solution
Utilize haploid inducing lines to deliver gene editing machinery via pollen, allowing simultaneous haploid induction and targeted mutagenesis without tissue culture, ensuring edited haploid progeny are produced and can be further doubled to create elite breeding lines.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If transformation methods (Agrobacterium or biolistic) are used to introduce editing machinery into plants, then gene editing can be achieved, but the method will not work for transformation-recalcitrant crops or varieties
Solution Approach 1:
The patent uses pollen as an intermediary vector to deliver editing machinery (Cas9 and guide RNA) to the target plant. Instead of directly transforming recalcitrant plant tissues, the editing components are introduced into the pollen of a compatible donor plant, which then naturally transfers them during pollination. This bypasses the transformation recalcitrance issue while achieving gene editing in the target plant.
Solution Approach 2:
The patent replaces mechanical transformation methods (Agrobacterium infiltration, biolistic bombardment) with a biological delivery system using pollen. The natural pollination process substitutes for complex mechanical transformation protocols, making the method applicable to recalcitrant varieties that cannot be transformed by conventional means.
2Manufacturing precision
If trait introgression is used to introduce edits in recalcitrant varieties, then edits can be introduced, but the process is expensive, laborious, time-consuming and introduces genetic impurities due to linkage
Solution Approach 1:
The patent extracts only the essential editing components (Cas9 protein and guide RNA) from the donor plant's pollen, delivering them to the target plant without transferring the entire genomic background. This selective extraction avoids the genetic linkage problem where entire chromosomal segments including unwanted linked genes are transferred during traditional introgression breeding.
Solution Approach 2:
The editing machinery is pre-assembled and delivered in the pollen before fertilization occurs. The Cas9 and guide RNA are already complexed and ready to act on the target genome immediately upon entry into the ovule, eliminating the need for multiple generations of breeding required in traditional introgression to achieve the desired edit.
3Adaptability or versatility
If editing machinery is introduced transiently to growing plants without tissue culture, then recalcitrant varieties can be edited, but it is challenging to ensure edits end up in cells that contribute to the germ-line
Solution Approach 1:
The editing machinery is delivered to the plant at the earliest possible stage - during pollination and fertilization - before the plant develops further. By introducing Cas9 and guide RNA into the zygote or early embryonic cells at this critical window, the patent ensures that the editing occurs in cells that will contribute to the germ line, enabling reliable transmission of edits to progeny.
Solution Approach 2:
Pollen serves as a natural intermediary that delivers editing machinery directly to the female reproductive tissues during pollination. This biological mediator ensures precise targeting of the germ line, as pollen naturally travels to and enters the ovule, delivering its cargo (including the editing components) directly to the cells that will form the embryo and future germ line.
4Reliability
If tissue culture is used for transformation, then stable transformation can be achieved, but the process is genotype-dependent and will not work for all crops or varieties
Solution Approach 1:
The patent replaces the mechanical and biochemical transformation system (Agrobacterium or biolistic methods requiring tissue culture) with a simple biological delivery system using pollen. Pollen-mediated delivery is a natural process that works across compatible species and varieties without requiring the target plant to be amenable to in vitro transformation, thus achieving both stability and broad versatility.
Solution Approach 2:
The patent creates a universal delivery system using pollen that can function across different crop species and varieties. Pollen is a universal biological vector for gene transfer in sexually reproducing plants, making this approach broadly applicable to recalcitrant varieties that cannot be transformed by species-specific tissue culture methods.
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. The disclosure is also directed to methods of testing an edited haploid plant progeny for the presence of a first plant's genomic material.